Relieving Allosteric Inhibition by Designing Active Inclusion Bodies

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Relieving Allosteric Inhibition by Designing Active Inclusion Bodies and Coating of the Inclusion Bodies with FeO Nanomaterials for Sustainable 2-Oxobutyric Acid Production 3

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Junping Zhou, Rong-Zhen Zhang, Taowei Yang, Qiaoli Liu, Junxian Zheng, Fang Wang, Fei Liu, Meijuan Xu, Xian Zhang, and Zhiming Rao ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03181 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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ACS Catalysis

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Relieving Allosteric Inhibition by Designing Active Inclusion

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Bodies and Coating of the Inclusion Bodies with Fe3O4

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Nanomaterials for Sustainable 2-Oxobutyric Acid Production

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Junping Zhou,†,‡ Rongzhen Zhang,*,†,‡ Taowei Yang,†,‡ Qiaoli Liu,†,‡ Junxian

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Zheng,†,‡ Fang Wang,†,‡ Fei Liu,†,‡ Meijuan Xu,†,‡ Xian Zhang,†,‡ Zhiming

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Rao*,†,‡

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†

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Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

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*Corresponding

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[email protected].

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Tel: +86-510-85197760; Fax: +86-510-85864112

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‡

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P. R. China

authors:

Rongzhen

Zhang,

[email protected];

Zhiming

Rao,

Present address: School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122,

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ACS Paragon Plus Environment

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Abstract

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Allosteric inhibition of key enzymes by end-product has been widely studied in

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control of valuable compound biosynthesis, and site-directed mutagenesis is often

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applied for relieving allosteric inhibition. Here we rationally designed a different

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approach to relieve allosteric inhibition by Ile in threonine deaminase (TD) pathway

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and efficiently produced 2-oxobutyric acid in a save-energy way. We truncated the

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different peptide length at C-terminal regulatory domain (from residue 11 to 193)

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Escherichia coli TD and obtained the active inclusion bodies. The truncated residues

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Glu480-Gly514 in C-terminal regulatory domain were confirmed to be an unstable

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structure by MD simulations. The truncation variants ∆11, ∆25 and ∆35 weren’t

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allosterically regulated by Ile at all by kinetics analysis. They showed decreased

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activity but better thermostability than wild-type enzyme. Among the three variants,

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∆25 showed 3.3-fold higher 2-oxobutyric acid production in the presence of 2 mM Ile

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and about 9-fold higher production at 55 °C than the wild-type. The inclusion bodies

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resulted from the aggregation of truncated TD dimers by hydrophobic interaction,

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could resist the binding of Ile from calorimetry experiments. According to attenuated

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total reflection FTIR measurement, the inclusion bodies kept a similar secondary

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structure composition as wild-type enzyme. Furthermore, by nonspecific adsorption

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of Fe3O4 nanoparticles onto inclusion bodies, they could be quickly recycled without

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enzyme leakage using save-energy magnetic separation. Since many allosteric

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enzymes show the similar structure as TD, our work provides a general and effective

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strategy for relieving enzymatic allosteric inhibition by end-products, and realizes

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sustainable chemical production using Fe3O4 nanoparticles for inclusion body

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immobilization.

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Allosteric

regulation,

threonine

deaminase,

42

Keywords:

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immobilization, magnetic nanoparticles, biocatalysis, reusability

inclusion

body,

44

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ACS Catalysis

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Introduction

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Allosteric regulation is used as a very efficient mechanism to control protein

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activity in most biological processes, including signal transduction, metabolism,

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catalysis and gene regulation. Allosteric regulation of key enzymes exists in various

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metabolic control of biosynthetic pathways,1 and the allosteric enzymes subject to

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feedback inhibition by the end-products of the pathway are usually present at the first

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committed step, thus down modulating the biosynthesis of the end-products.2-3

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Acetolactate synthase, the first enzyme dedicated to the valine biosynthetic pathway,

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shows feedback inhibition by end-products Val and Ile in plants.2 Glutamine

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5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase which catalyzes the first

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committed step in de novo biosynthesis of purine nucleotides, also shows feedback

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inhibition by adenosine and guanosine mono- and diphosphate end-products of purine

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biosynthesis in bacteria.4 The mechanism of allosteric enzymes subject to feedback

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inhibition differs from that of the product inhibition during catalysis.5 The

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conformational mobility between allosteric regulation and catalysis is usually

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discussed as the common route.6-7 However, lots of work remains to be done to

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explore new allosteric enzymes and fully understand these principles of allosteric

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regulation.

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To desensitize key enzymes from end-products inhibition, many works focus on

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random mutation and selection, such as selection of α-amino-β-hydroxylvaleric acid

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(AHV, a threonine analog) resistant variants used to relieve the threonine allosteric

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inhibition of homoserine dehydrogenase for threonine biosynthesis.8-10 However, this

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approach is time-consuming and requires huge input to explore the useful sites or

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select the positive variants for relieving the end-products allosteric inhibition.

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Immobilization can be another solution for various inhibition problems, usually on

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account of steric exclusion of the substrate from an ‘‘inhibition site’’ or rigidification

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of the enzyme structure by multipoint covalent immobilization.11-13 Penicillin G

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acylase is found to be less allosteric inhibited by the nucleophile after multipoint

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covalent immobilization using CL-6-glyoxyl agarose.14 Since immobilizations are

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usually conducted in vitro to diminish enzyme inhibition while allosteric inhibitions

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happen in vivo for various metabolic control of biosynthetic pathways, it is more

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necessary to find a way of relieving allosteric inhibition by engineering the enzyme

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itself. A classic example could be provided using biosynthetic L-threonine deaminase

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(TD, EC 4.2.1.16), an important enzyme which catalyzes the deamination of threonine

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into 2-oxobutyrate acid (2-OBA) in the isoleucine biosynthetic pathways, as also

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sensitive to Ile inhibition. The other type of L-threonine deaminase is catabolic

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enzyme that is not inhibited by Ile. This catabolic enzyme is only produced when the

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E. coli cell grown anaerobically in a medium containing high concentrated amino

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acids and without glucose.15-16 By studying the Ile inhibition, it is reported that the

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identical subunits of TD are organized into two distinct domains, a catalytic N

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terminal domain containing the pyridoxal phosphate (PLP) cofactor and a regulatory

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C-terminal domain.17 Isoleucine was shown to be an allosteric inhibitor by decreasing

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the affinity for threonine, whereas valine reversed this inhibition.8-11 The regulatory

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domain of each monomer possesses two effector-binding sites constituted in part by

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Y449 and Y543 are studied, the latter is responsible for conformational modifications

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leading to the final inhibition of the enzyme while Y449 interacts with both isoleucine

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and valine.18 However, interaction of valine with the high-affinity binding site

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induced different conformational modifications, leading to the dissociation of

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isoleucine from binding sites and the reversion of inhibition.18 Information about the

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binding sites and the regulation mechanisms of the effectors Ile and Val in TD of E.

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coli has also been studied in detail.16 But only a limited amount of TD variants have

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been identified to desensitize TD from Ile inhibition, all of which were exclusively

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identified by random mutation and selection.9, 16 Therefore, it is important to find a

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robust simpler method for relieving end-product allosteric inhibition.

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As most of the key enzymes with end-product allosteric inhibition have their

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regulatory domains, it is reasonable to take their regulatory domains into account for

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modification. C-terminal of TD is important because of its role in regulation of Ile and

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Val metabolism. Therefore we designed truncated EcTD in regulatory C-terminal

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domain, and several truncated TDs turned to be active inclusion bodies which relieved

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the allosteric inhibition by Ile and they performed better thermostability. By exploring

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of this supermolecular bio-device inclusion body immobilization, we found that Fe3O4

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could be a suitable material. Fe3O4 has been widely applied for enzyme

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immobilization as magnetically recoverable catalysts because of its low toxicity and

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biocompatibility.19-20 The successful coating of TD inclusion bodies with Fe3O4

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nanomaterials was confirmed by infrared spectroscopy (IR) and transmission electron

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microscopy (TEM). The composite could produce 2-OBA, an important precursor for

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chiral 2-aminobutyric acid,21 with save-energy enzyme recovery by magnetic

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separation while no leakage of enzymes from the composite happened.

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Results and discussion

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Design active inclusion bodies by the truncation of TD C-terminal peptides. TD is

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a rate-limiting enzyme in isoleucine synthesis pathway in microorganisms. It is

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allosterically inhibited by Ile.8-11 The crystal structure of E. coli TD is a tetramer,

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which can be described as a “dimer of dimers”. Most of the interactions take place in

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the C-terminal regulatory domain of each dimer, not in the N–terminal catalytic

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domains of the dimers.22 In this work, we rationally designed the regulatory domain

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of TD basing on an enzyme simplification project. We truncated different lengths of

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the C-terminal peptides at regulatory domain (from residue 11 to 193) of E. coli TD.

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The wild type (WT), ∆42 and ∆193 enzymes were expressed as soluble proteins in E.

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coli BL21, while the truncation variants ∆11, ∆25 and ∆35 were produced as inclusion

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bodies (see Figure S1). The WT enzyme presented 47.8±2.0 µmol mg-1 min-1, while

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the truncation variants ∆11, ∆25 and ∆35 showed 15.5±0.9, 17.9±1.0 and 16.2±0.9

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µmol mg-1 min-1 in the 2-OBA production, respectively. Compared to WT, the variants

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∆11, ∆25 and ∆35 presented about 3-fold decrease activity. The truncation variants

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∆11, ∆25 and ∆35 showed lower activities towards 2-OBA production due to the

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formation of inclusion bodies. The variants ∆42 and ∆193 showed no any activities,

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suggesting they were lethal mutations. The main reason was the overtruncation of

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C-terminal regulatory domain or TD.

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To analyze the formation of inclusion body by the truncation of C-terminal

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peptides, we modeled the structures of TD truncation variants. As shown in Figure 1,

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the WT enzyme structure (PDB ID: 1TDJ) contains two domains: the N–terminal

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catalytic domain and the C-terminal regulatory domain. The β-sheets s15, s17, s16

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and s12 formed a closed structure with the α-helix h18 of C-terminal of WT enzyme.

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The E480-G514 peptide in C-terminal of WT enzyme was confirmed to be an

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unstable structure by the study of root-mean-square fluctuations (RMSFs) according

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to the MD simulations (labelled in Figure 1B). And the overtruncation variants such

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as ∆42 and ∆193 involved the rigid structure in C-terminal of WT enzyme might

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result in the collapse of the TD enzyme structure, thus causing the loss of enzymatic

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catalytic function. However, the C-terminal truncation ∆11, ∆25 and ∆35 led to

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exposure of four β-sheets (s15-RLYSFE, s17-VLAAF, s16-LFH, partial of

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s12-SVTEFN) with all sites being enriched in hydrophobic and aromatic residues to

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the solvent (labelled in Figure 1C). It is reported that SecB-recognition sites in PhoA

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and in MBP are also enriched in hydrophobic and aromatic residues, and with the help

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of these residues, SecB uses long hydrophobic grooves that run around its disk-like

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shape to recognize and bind to multiple hydrophobic segments across the length of

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non-native proteins.23 Thus, based on similar principles, the exposure of the

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hydrophobic residues, such as L435, L476, A477, A478 and L462 in the β-sheets

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easily caused homodimer aggregation due to the hydrophobic interactions, and thus

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the formation of inclusion bodies happened with many truncated TD homodimers

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buried inside (Figure 1D). Recently, active inclusion bodies achieved by fusion of

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self-assembling peptide to the carboxyl termini of proteins are formed because of

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peptide-induced aggregation,24 this could also help to explain the formation of

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inclusion bodies ∆11, ∆25 and ∆35. As shown in Figure 1E, the inclusion bodies

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distributed uniformly with the diameter ranging from 100 to 500 nm. It was worth

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mentioning that the truncation of C-terminal domains had no effect on the structure of

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N terminal catalytic domain, which remained hydrophilic chain. The hydrophilic

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N-terminal catalytic domain of inclusion bodies exposed to the solution as the surface

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of inclusion bodies, where maintained the original protein structure and showed

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enzymatic activities (shown in Figure 1D). However, these inclusion bodies showed

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lower activities than WT enzyme. The possible reason was that the substrate could

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only reach the surface of inclusion bodies.

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Insert Figure 1

167 168

Kinetics and ITC experiments demonstrated that the truncation variants weren’t

169

inhibited allosterically by Ile. To determine whether the truncation variants were

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allosterically inhibited by Ile, the steady-state kinetics of WT enzyme and its variants

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∆11, ∆25 and ∆35 were conducted. The fitted data based on the Hill equation was

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shown in Table S2. The kinetics of WT showed that Ile increased the K0.5 value of Thr

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from 18.38±2.39 mM to 25.12±4.60 mM with no effector, while Val slightly

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decreased the K0.5 value of Thr to 15.71±0.69 mM (Figure 2A). These results were

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consistent with previous reports that Ile decreases the affinity of Thr whereas Val

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increases the affinity of threonine.18, 25-26 For the truncation variants of TD, Ile could

8


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ACS Catalysis

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not decrease the affinity between enzyme and Thr, no matter whatever concentration

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of Ile was added, suggesting that these truncation variants of TD relieved the

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allosteric regulation of Ile. For the WT enzyme, a concerted addition of Ile and

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appropriate concentration of Val (10-fold Ile) could evidently reduce the K0.5 values of

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Thr compared to that with the only addition of Ile, suggesting Val could reverse the Ile

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inhibition partially for the WT enzyme. However, the TD inclusion bodies were not

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affected by Val (Figure 2A).

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To understand the reason of relieving Ile allosteric inhibition in truncated TD

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variants, we performed isothermal titration calorimetry (ITC) measurements on

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binding affinity of Ile with enzymes. Figure 2B showed raw data when Ile was added

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to WT enzyme solution and the integrated heat change plotted against molar ratio. The

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resulting thermogram was well described by a model for identical binding sites with a

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Kd of 5.41 ± 2.07 µM, ∆H of -59.5 ± 9.7 kcal mol-1, -T∆S of 29.4 kcal mol-1, and

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number of sites per monomer (n) of 0.80 ± 0.05. The binding of Ile on WT enzyme

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was driven by enthalpy change and the hydrogen bond was formed, following the

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conformation change to allosteric inhibition. These results were in agreement with

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reported mechanism of the allosteric regulation of TD.16 However, the ITC results

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indicated that no binding happened between the Ile and truncated TD variants (Figure

195

S2), suggesting that the formation of TD inclusion bodies prevented the accession of

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regulator Ile.

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Insert Figure 2

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Ile almost didn’t affect 2-OBA production. As shown in Figure 3A, WT enzyme

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showed IC0.5 (inhibition) 0.34±0.03 mM, indicating that Ile strongly inhibits the

201

enzyme. 25 However, Ile had no influence on the activities of the truncation variants of

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TD even with 10 mM Ile, corresponding to the above results shown in Figure 2. The

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biotransformation showed that WT enzyme produced 86.8±2.8 g L-1 2-OBA without

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the addition of Ile, while it produced 2-OBA with a low yield of 19.0±0.2 g L-1 when

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1 M Thr was added to the reaction system in the presence of 2 mM Ile (Figure 3B).

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The truncation variant ∆25 showed almost the same 2-OBA production in either the

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presence or absence of 2 mM Ile. These results further confirmed that the appropriate

208

truncation at the C-terminal regulatory domain relieved the allosteric inhibition of Ile

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in TD pathway.

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Attenuated total reflectance FTIR has been used to study the secondary structure

211

and conformation changes of protein, such as the human interleukin-1β in inclusion

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bodies and other aggregated forms,27 heat-induced denaturation of defatted bovine

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serum albumin.28 Figure 3C showed the curve fitting results for the deconvolved IR

214

spectra of WT enzyme and its truncated variants. For WT enzyme, seven bands were

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identified at 1685, 1669, 1649, 1646, 1637, 1629 and 1609 cm-1, which were assigned

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to

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β-sheet/extended structures, β-sheet/extended structures, and vibration od some amino

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acid residues.27-28 The IR spectrums of ∆11 (red dashed line) and ∆35 (pink dashed

219

line) were almost the same as that of ∆25 (black solid line), so only ∆25 was

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estimated by curve fitting procedures. There were five bands for ∆25 IR spectrum

intermolecular

β-sheet

structures,

turn

structures,

a-helical

structures,

10


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identified at 1683, 1653, 1636, 1625 and 1615 cm-1. The secondary structure of native

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WT enzyme was composed of 43.9±1.2% a-helix, 24.9±0.8% β-sheet and 28.5±0.6%

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turn. This showed some similarity with 37.5% a-helix and 17.3% β-sheet of native

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WT enzyme determined by X-ray crystallography, and the differences could be due to

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that the secondary structure deduced by FTIR usually reveals some difference with

226

that determined by X-ray crystallography or NMR.27, 29 The secondary structure of

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∆25 was composed of 44.7±3.9% a-helix, 24.2±1.7% β-sheet, 15.8±1.2% turn and

228

15.2±0.5% intermolecular β-sheet. The almost the same secondary structure between

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WT enzyme and ∆25 indicated that inclusion bodies might have functional dimer

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structure as WT enzyme, except that the 15.2% intermolecular β-sheet in ∆25 resulted

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from the aggregation of functional dimers. Formation of the intermolecular β-sheet

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structures can be with very strong hydrogen bond interactions leading to irreversible

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aggregation of the protein,28 while this structure could also prevent the accession of

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Ile into the regulatory domain and relieving the allosteric inhibition in the inclusion

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bodies of truncation variants.

236

MD simulations under a constant temperature of 30°C showed that differences in

237

the RMSFs (∆RMSF) of the WT enzyme and truncated variants were small except the

238

E480-G514 peptide in C-terminal of WT enzyme (Figure 3D). These results proved

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that the monomer of TD enzyme could keep a stable conformation even with the

240

truncation of E480-G514 peptide in C-terminal regulatory domain. The monomers

241

from variants ∆11, ∆25 and ∆35 also showed similar radius of gyrations (Rg) as that

242

of WT enzyme (Figure S3), suggesting that the monomers from variants ∆11, ∆25 and

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∆35 showed similar tertiary structures as monomer of WT enzyme, which was

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reported by Lobanov et al. They thought that the protein radius of gyration

245

normalized by the radius of gyration of a ball with the same volume is independent of

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the protein size, in contrast to compactness and the number of contacts per residue.30

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Allosteric regulation mechanism is often used to control enzyme activity in most

248

biological processes, which includes signal transduction, metabolism, catalysis and

249

gene regulation.31-32 Comparing with the normal techniques of site-directed mutation,9,

250

16

251

the truncation in regulatory domain would not affect the catalytic function of TD. The

252

structure of TD can be divided into regulatory part and catalytic part which are

253

connected by a thin neck-like region.16 However, the strategy might be not suitable for

254

enzymes with closely associated regulatory and catalytic domains. More interestingly,

255

due to the necessity of allosteric regulation, regulatory domains are usually far apart

256

from the catalytic domains, binding of effector molecules to allosteric sites modulates

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structural dynamics, thus affecting activity of remote functional sites.33 Many

258

allosteric proteins show a similar structure as TD, such as catabolite activator

259

protein,32 human seven sirtuins,34 nitrogen regulatory protein C and so on,31, 35-36.

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Therefore, our strategy could be potentially applied for these proteins to change the in

261

vivo metabolism.

262

we provided a much simpler way to relieve end-product allosteric inhibition since

Insert Figure 3

263 264

The coating of truncation variants with Fe3O4 nanomaterials enhanced the

12


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catalytic functions. Although the truncation variants of TD relieved the allosteric

266

inhibition, they were expressed as inclusion bodies and showed lower activity than the

267

WT enzyme. In order to improve the catalytic function of truncation variants, we

268

attempted to design a nanoparticle to immobilize them. Magnetic separation generally

269

offers high efficiency and specificity when compared with equivalent centrifugation

270

or filtration methods.37 However, the successful bio-conjugation of proteins and

271

magnetic materials is usually based on the decoration of magnetic materials with

272

polyamidoamine,38

273

3-methacryloxypropyltrimethoxysilane or some other chemicals.40 Here, the Fe3O4

274

prepared by hydrothermal process without decoration was found to be a good material

275

for inclusion body coating. It was reported that Fe3O4 nanomaterials could be an

276

excellent material by fast physical adsorption and this was confirmed by IR. As

277

depicted in the XRD patterns of Fe3O4 nanomaterials (shown in Figure S4A), five

278

characteristic diffraction peaks of Fe3O4 were corresponding to the cubic phase of

279

Fe3O4 (JCPDS 74-0748).41 SEM image (Figure S4B and S4C) showed that the

280

particle sizes of Fe3O4 were nanoscale with the particle diameter below 100 nm.

carboxymethylated

chitosan,39

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As shown in Figure 4A, IR spectrum of the inclusion body ∆25, the nanoparticle

282

Fe3O4 and the coating of ∆25 inclusion bodies with Fe3O4 composite (Fe3O4/∆25) was

283

analyzed. Based on detecting the peptide bonds, 1635 cm-1 revealed the stretching

284

vibration of C=O in peptide bond which is weaker than that of C=O in aldehyde or

285

ketone group because of the mesomeric effect.42-43 1523 cm-1 revealed the stretching

286

vibration of N-H in peptide bond with a major transconfiguration structure.42 The

13


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small adsorption at the peaks of 1450 cm-1 and 1380 cm-1 were due to the bending

288

vibration of methyl or methylene.44 The strong adsorption peaks of about 900-1000

289

cm-1 similar to IR spectrum of Fe3O4 usually resulted from the vibration of

290

metal-oxygen double bonds.45 These peaks changes existed in the IR spectrum of ∆25

291

and the Fe3O4/∆25 composite, confirming that the inclusion body of ∆25 was coated

292

by Fe3O4 nanomaterials. Figure 4B showed the TEM and schematic diagrams of ∆25,

293

Fe3O4/∆25 and the nanoparticle Fe3O4. It revealed that agglomerated Fe3O4

294

nanoparticles were adsorbed nonspecifically around the ∆25 inclusion bodies.

295

Insert Figure 4

296 297

The Fe3O4/∆25 composites were prepared by mixing Fe3O4 nanomaterials with

298

the enzymatic solution coupled with stirring for 10 minutes at 4 oC. The capacity for

299

TD inclusion body coating by Fe3O4 nanomaterials was detected at different pH

300

(Figure 5). The highest capacity of immobilized ∆25 inclusion bodies was 1.35±0.07

301

mg mg-1 Fe3O4 in PBS buffer (pH 7.0), while the immobilized capacity at pH 5.5 or

302

pH 9.5 was only about 0.75 mg mg-1. In most cases, high and low pH values are

303

adverse for adsorption, as high pH often causes electrostatic repulsion between the

304

negatively charged adsorbent and adsorbate, while low pH often causes electrostatic

305

repulsion between the positively charged adsorbent and adsorbate.46-47 However,

306

Tatsumi et al. studied the adsorption amount of horseradish peroxidase on magnetite

307

and found that usually a small amount (0.003 mg mg-1) of soluble protein can be

308

absorbed on magnetite.48 Differed from their soluble proteins, our active TD inclusion

14


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ACS Catalysis

309

bodies could be termed as single-enzyme supramolecular devices with bigger

310

molecular mass, and it is well known that Van der Waals force generally increases

311

with the increase of relative molecular mass.49 Thus Van der Waals force between

312

Fe3O4 and active TD inclusion bodies could be much stronger, resulting in a higher

313

amount for adsorption. Also such a fast velocity of immobilization is usually existed

314

during the physical adsorption for protein immobilization,12,

315

nanomaterials could easily be used for industrial immobilization of inclusion bodies

316

by coating. The HRTEM picture (Figure 5C) revealed that crystalline Fe3O4

317

nanoparticles shared interface with the unsmooth surface of inclusion bodies.

318

Moreover, as the inclusion bodies were expressed with 6×histidine-tagged protein, the

319

effect of histidine-tag in immobilization was determined by the addition of high

320

concentrated imidazole into the Fe3O4/∆25 composites. We found that the ∆25

321

inclusion bodies did not release from the nanomaterials even with 1 M imidazole,

322

suggesting that the main interactions between Fe3O4 and ∆25 was not caused by

323

histidine-tag or electrostatic adsorption.

50

and Fe3O4

324

The effects of the ion strength, mild solvents and nonionic surfactant on the

325

adsorption efficiency and enzyme activity of inclusion bodies were shown in Figure

326

S5. It was reported that the ion strength increase can enhance hydrophobic

327

interaction.51-52 In this work, it resulted to a decreased adsorption rate, indicating that

328

hydrophobic interaction was not the major interaction force in Fe3O4/∆25 composites.

329

It is reported that Fe can interact with the N atom of -NH2 by experiments and MD

330

simulations,53-56 which indicated that ionic exchange interactions could be an

15


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 47

331

interaction force in Fe3O4/∆25 composites. Moreover, the decreased adsorption rate

332

under a high ion strength for Fe3O4/∆25 composites might also be due to the formed

333

outer-sphere surface complex (OSSC) between Fe3O4 nanoparticles and inclusion

334

bodies.57-58 OSSC is usually developed by weak non chemical bonding force, such as

335

hydrogen bond, electrostatic and van der Waals force.59 It is reported that Fe3O4

336

nanoparticles prepared in the aqueous phase are covered with a number of hydroxy

337

(-OH) groups,60-61 which was attribute to the hydrogen bond between inclusion bodies

338

and Fe3O4 which has been proved by MD simulations and the radial distribution

339

function analysis between Fe3O4 and biocompatible polymer.56 This explained that

340

addition of glycerin or PEG4000 or nonionic surfactant F127 all caused obvious

341

decreased adsorption rate of Fe3O4 nanoparticles on inclusion bodies in our study,

342

because these chemicals had abundant hydroxyl and might competitive bind on the

343

surface of Fe3O4 nanoparticles due to the hydrogen bond. Thus, the major interaction

344

forces between Fe3O4 nanoparticles and inclusion bodies were hydrogen bond and

345

ionic exchange interaction forces, while van der Waals force might also play a role.

346

Insert Figure 5

347 348

The biotransformation of 2-OBA by WT, ∆25 and Fe3O4/∆25 composites were

349

conducted at different pH and temperature. The results were summarized in Figure 6.

350

WT produced 102.8±6.8 g L-1 2-OBA at pH 9.0, and about 75 g L-1 at pH 6.0-7.0 in

351

8 h. The molar conversion of Thr for WT enzyme at pH 9.0 was 99.8±6.6%. The

352

variant ∆25 showed the highest yield 79.5±5.4 g L-1 of 2-OBA under neutral

16


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ACS Catalysis

353

conditions, and declined 2-OBA production under alkaline conditions, which

354

resulted from the truncation of residues at C-terminal regulation domain. The

355

Fe3O4/∆25 composites produced the similar yield of 2-OBA as ∆25 under neutral or

356

alkaline conditions. Quite surprisingly, the Fe3O4/∆25 composites showed very low

357

2-OBA yield (2.4±0.3 g L-1) under acidic conditions, an enormous difference from

358

that of ∆25. By checking the WT enzymatic properties, we found that the WT

359

activity could be completely inhibited by 1 mM Cu2+ or Fe3+ (Figure S6). Under

360

acidic conditions, Fe3O4 can be dissolved and thus Fe3+ is relieved in solution.62

361

Although the truncated TD C-terminal could relieve the inhibition of the end-product

362

Ile, it could not relieve the inhibition of Fe3+ probably because the binding site of

363

Fe3+ or other cations with TD is speculated to N-terminal catalytic domain.18

364

With the formation of active inclusion bodies, the ∆25 showed improved

365

thermostability. WT produced about 75 g L-1 2-OBA at temperature below 45 oC,

366

49.0±0.9 g L-1 2-OBA at 50 oC, and only 6.5±0.4 g L-1 2-OBA at 55 oC. With the

367

increasing temperature, the 2-OBA production was decreased due to the unstable

368

enzyme. The ∆25 produced 51.0±1.5 g L-1 2-OBA at 55 oC, which was 6.8 times

369

higher production than WT. The ∆25 yielded 22.5±1.2 g L-1 2-OBA, whereas the WT

370

could not produce 2-OBA at 60 oC. The Fe3O4/∆25 produced 60.2±1.5 g L-1 2-OBA

371

at 55 oC, and 24.9±0.9 g L-1 2-OBA at 60 oC, a bit higher than ∆25. The higher

372

thermostability of inclusion bodies resulted from the aggregation of truncated TD

373

dimers by hydrophobic interaction, as reported by Rinas et al. that the inclusion

374

bodies can act as mechanically and functionally stable porous catalysts and are more

17


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375 376

Page 18 of 47

resistant to thermal inactivation than the soluble version of the enzyme.63-64 Insert Figure 6

377 378

Sustainable production of 2-OBA by the Fe3O4/∆ ∆25 composites. The inclusion

379

bodies of ∆25 can be separated by centrifugation from the reaction mixture. However,

380

the amount of enzymes was diminished drastically after several recycle times which

381

could be due to the leakage of inclusion bodies in supernatant (Figure 7). The

382

Fe3O4/∆25 composites could be simply separated using magnet, therefore the product

383

was easily purified (Figure 7A). The Fe3O4/∆25 composites produced 7.12±0.26 g L-1

384

h-1 2-OBA after being recycled 20 times. The production was about 72% of initial

385

production. The ∆25 produced only 2.28±0.07 g L-1 h-1 2-OBA after 20 times, which

386

was about 23% of its initial production. The protein concentration of ∆25 solution was

387

0.090±0.005 mg mL-1 after 20 times recycles, which was approximately 31% of its

388

initial protein concentration, while there was almost no leakage of enzymes using

389

Fe3O4/∆25 composites after 20 times recycles. These results suggested that leakage of

390

inclusion bodies occurred during the centrifugation. The concentration of Fe3O4/∆25

391

composites solution remained almost the same after 20 times recycle (Figure S7),

392

which could prove that enzyme desorption did not occur for Fe3O4/∆25 composites

393

even after 20 times transformation.

394

The Fe3O4 was an excellent material for the coating of inclusion bodies, a type of

395

supramolecular bio-devices. This type of immobilization obviated the use of harsh

396

ligation conditions that usually denatured fragile proteins. Advantages of enzyme

18


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ACS Catalysis

397

immobilization using magnetic nanoparticles include enhancement of thermostability

398

65

399

possibility of acting on large even solid substrates for enzyme.68 Althoug most of this

400

immobilization are achieved by glutaraldehyde crosslinking with long reaction time,

401

the process in our study was finished in a very short time based on physical

402

adsorption. Immobilization of proteins using such supramolecular noncovalent

403

interactions has great potential in biomolecular and biotechnological research.69 Since

404

Jean-Marie Lehn proposed the conception of supramolecular chemistry, aiming at

405

developing highly complex chemical systems from components interacting by

406

noncovalent intermolecular forces,70 and it offers diverse opportunities for fabrication

407

and improvement of bio-devices. At the same time, as one type of supramolecular

408

bio-devices, multienzyme supramolecular bio-devices including our inclusion bodies

409

has been widely applied in cell-free biosystems for biochemical and biofuel

410

production.71-72 It has been reported that multienzyme supramolecular bio-devices can

411

be constructed using oligomer-to-multimer strategies by taking advantage of the

412

cooperation of metal-ion-chelating interactions and nonspecific protein-protein

413

interactions,73 enzyme fusion with a ligand,74 enzyme polymerization induced by

414

small molecules and so on.75-76 Nature utilizes template-directed assembly to

415

construct well-defined biopolymers such as nucleic acid and proteins, these have

416

inspired chemists to synthesize artificial receptors and other complex architectures

417

such as interlocked molecules.77-78

418

supramolecular bio-devices could get a wider application in catalyst or other fields.

and biocatalytic activity,66 the higher enantioselectivity,67 good reusability and

With the help of Fe3O4 nanomaterial, these

19


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419

Page 20 of 47

Insert Figure 7

420 421

Conclusion

422

In summary, we designed the active inclusion bodies by truncation at C-terminal

423

regulation domain to relieve the allosteric inhibition of end-product Ile in TD pathway.

424

These inclusion bodies showed the higher thermostability and then was used for

425

production of 2-OBA, an important intermediate for L-2-aminobutyric acid and

426

isoleucine production. As many allosteric proteins showed a similar structure as the

427

TD with regulatory domains far apart from the catalytic domains, our work provided

428

an effective strategy for relieving enzymatic allosteric inhibition by end-products in

429

biosynthetic pathways. To improve the catalytic function, the Fe3O4 composites were

430

designed for the coating of inclusion bodies. The Fe3O4/∆25 composites significantly

431

enhanced 2-OBA production. Even after being recycled for 20 times, the yield was

432

maintained about 72%. More importantly, the Fe3O4/∆25 had enabled recycling using

433

magnet separation method and the product 2-OBA was easily separated from the

434

reaction mixture. This work realized the efficient and sustainable 2-OBA production

435

by the truncation variant of TD to relieve the allosteric inhibition of Ile, and Fe3O4

436

nanomaterial for active inclusion body coating to be recycled. It supplies an easy way

437

with potential industrial application for separation of supramolecular bio-devices

438

including inclusion bodies from the reaction mixture and eventual recycle.

439 440

Materials and Methods 20


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ACS Catalysis

441

Microorganisms and chemicals. E. coli str. K-12 substr. MG1655 was used as donor

442

of L-threonine deaminase gene ivlA (Gene ID: 948287). E. coli JM109 and E. coli

443

BL21 (DE3) were used as host for gene cloning and expression, respectively. The

444

vector pMD18-T (TaKaRa Co., Japan) was used as cloning plasmid. The plasmid

445

pET-28a (Novagen Co., Darmstadt, Germany) was used for gene expression.

446

L-threonine, L-isoleucine and L-valine were purchased from Sinopharm Chemical

447

Reagent Co., Ltd (Shanghai, China). The 2-oxobutyric acid was purchased from

448

Sigma-Aldrich

449

pyridoxal-5-phosphate hydrate (PLP) was purchased from Sangon Biotech Co., Ltd

450

(Shanghai, China). The restriction enzymes, Extaq DNA Polymerase and T4 DNA

451

ligase were purchased from TaKaRa Bio. Co. (Dalian, China). All other chemicals of

452

high grade were obtained from commercial source.

453

Construction of truncated TD gene. The ilvA gene and its truncated sequence at its

454

C-terminal were cloned using the genome of E. coli str. K-12 substr. MG1655 as

455

template. Their BamH I-Hind III fragments were cloned into the corresponding sites

456

of plasmid pET-28a. The recombinant plasmids pET28a-TD, pET28a-∆11,

457

pET28a-∆25, pET28a-∆35, pET28a-∆42 and pET28a-∆193 were transformed to the

458

competent E. coli BL21, and the recombinant strains E. coli BL21/pET28a-TD, E.

459

coli BL21/pET28a-∆11, E. coli BL21/pET28a-∆25, E. coli BL21/pET28a-∆35, E. coli

460

BL21/pET28a-∆42 and E. coli BL21/pET28a-∆193 were obtained. All the positive

461

clones were verified by DNA sequencing. The primers used in this work were listed in

462

Table S1 in the supporting information.

(St.

Louis,

USA).

Isoropyl-β-D-thiogalactoside

(IPTG)

and

21


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Page 22 of 47

463

Expression and purification of TD and its variants. All recombinant proteins were

464

expressed in E. coli BL21 (DE3) as His6-tagged proteins. All the recombinant E. coli

465

strains were cultured in LB medium containing 50 µg ml−1 kanamycin at 37 °C. When

466

OD600 value of the culture reached 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG)

467

was added to induce protein expression with a final concentration of 1 mM. The

468

cultures were cultivated at 28 °C for 8 h and then harvested by centrifugation (8,000 ×

469

g, 5 min).

470

The harvested cells were resuspended in 50 mM PBS buffer (pH 7.5) and lysed

471

by sonication with an ultrasonic oscillator (Sonic Materials Co., USA). The cell debris

472

were removed by centrifugation (12,000 × g, 40 min) at 4 °C, and the supernatant was

473

applied to a HisTrap HP affinity column (GE Healthcare, Piscataway, NJ, USA)

474

equilibrated with a buffer (20 mM Tris–HCl, 0.3 M NaCl; pH 7.5), and then was

475

eluted with a buffer (20 mM Tris–HCl, 0.3 M NaCl, 0.3 M imidazole; pH 7.5) using

476

an ÄKTA purifier system (GE Healthcare, Piscataway, USA). The homogeneity of

477

purified enzymes was determined by Coomassie brilliant blue staining of SDS-PAGE

478

gels. For recombinant E. coli with expressing TD inclusion bodies, cells were

479

disrupted by sonication followed by centrifugation (12,000 × g, 10 min) at 4 °C. Then

480

the pellet was washed up to 5 times with 50 mM PBS buffer (pH 7.5) and

481

resuspended in the same buffer. These TD inclusion bodies were not carried on for

482

further purification, because inclusion bodies are usually regarded as a relatively pure

483

source of recombinant protein which can be transformed into the active soluble form

484

by solubilization and subsequent refolding.63 The protein content was measured using

22


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ACS Catalysis

485

the Bradford assay.

486

Enzyme assay and kinetic determination. Enzymatic activities of TD and its variant

487

enzymes were measured at 30 °C by recording 2-OBA formation at absorbance value

488

230 nm after removing the background absorbance. One unit of enzyme activity is

489

defined as the amount of enzyme catalyzing the formation of 1 µmol 2-OBA per

490

minute under measurement conditions. Enzymatic activity was detected with 40 mM

491

threonine, a certain amount of enzyme, 20 µM PLP in 50 mM phosphate buffer (pH

492

8.0) at room temperature. The background absorbance was detected with the same

493

buffer with adding boiled enzyme, while the enzymatic activity of control was

494

detected using recombinant E. coli BL21/pET28a. The effect of Ile on the TD activity

495

was assayed at the presence of different concentrations of threonine (5 mM, 8 mM, 10

496

mM, 20 mM, 30 mM, 40 mM, 60 mM, 80 mM and 100 mM).

497

Kinetic parameters were measured and calculated using a Biotek Epoch2

498

spectrophotometer (Winooski, USA) equipped with a Gen5 software package and a

499

temperature control module. The absorbance was collected every 30 seconds and the

500

reaction would last for 4 minutes. Various concentrations of substrate threonine (5 to

501

100 mM), enzyme (3.4±0.1 µM), and 20 µM PLP in 50 mM phosphate buffer (pH 8.0)

502

were used. The effector was added to be 0.5 mM Val, 0.3 mM Ile or 0.3 mM Ile plus 3

503

mM Val in experiments. The K0.5 is defined as the corresponding concentration of

504

substrate Thr when the activity reaches 50 % of maximum rate. The reported values

505

represent the average of at least three independent measurements. The enzyme

506

kinetics was calculated by fitting the specific equation mentioned in results.

23


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 47

507

The inhibition of Ile on wild type or truncated TDs and affection of Ile for their

508

2-OBA production were also conducted. The Ile inhibition on enzymatic activities

509

was assayed as like kinetic parameter experiments in 50 mM phosphate buffer (pH 8.0)

510

in 96-well plates, with concentrations of Ile ranging from 0 to 10.0 mM (0 mM, 0.1

511

mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 2.0 mM, 4.0 mM, 6.0 mM, 8.0 mM and

512

10.0 mM). The effect of Ile on 2-OBA production was carried out using 1 M Thr in

513

the presence or absence of 2 mM Ile, the WT enzyme and truncated variant enzyme

514

concentration is 3.4±0.1 µM. The process for 2-OBA production lasted 8 hours at 30

515

o

516

adjusted to keep at 8.0 with 20% ammonia. 2-OBA was analyzed at 210 nm by

517

Agilent 1260 HPLC DAD detector (Agilent Technologies, USA) in ion

518

chromatographic column (Aminex HPX-87H, Bio-Rad, USA) using 5 mM H2SO4 as

519

mobile phase,16 the flow rate was 0.5 mL min-1 and the column temperature was kept

520

at 60 oC. Data were collected from three independent experiments with error bars

521

showed the standard deviation.

522

Isothermal titration calorimetry (ITC). ITC experiments were conducted on a

523

MicroCal PEAQ-ITC instrument (Malvern, UK). WT enzyme and truncated variants

524

(5 µM) in 50 mM sodium phosphate with pH 8.0 was loaded into the calorimeter cell,

525

and the titration syringe was loaded with 200 µM Ile in 50 mM sodium phosphate (pH

526

8.0). Titrations were carried out using 18 2-µL injections at 2-minute intervals at

527

25 °C. The data were fit with the equation for identical binding sites using MicroCal

528

PEAQ-ITC analysis software (Malvern, UK). The data in ITC were chosen from three

C in 50 mM phosphate buffer (pH 8.0). During the transformation, the pH was

24


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ACS Catalysis

529

independent experiments.

530

Preparation of Fe3O4 nanomaterials. The Fe3O4 nanoparticles were prepared

531

according to the method described by Goon et al..79 0.7 g of FeSO4 was dissolved in

532

80 mL distilled water, and 10 mL of 2.0 M KNO3 and 10 mL of 1.0 M NaOH were

533

added in an oxygen free environment. The initially formed Fe(OH)2 was heated at

534

90 °C for 2 h by Jinghong DK-S26 water bath (Shanghai, China), during which

535

Fe(OH)2 was oxidized to Fe3O4 nanoparticles. Particles were magnetically separated

536

from reaction mixture by placing a neodymium disk magnet (25 mm diameter) below

537

the reaction vessel (200 mL beaker) for 5 min to capture all magnetic particles before

538

discarding the reaction solution. The collected Fe3O4 particles were rinsed 5 times

539

with distilled water then dried in a Jinghong DZF-6050 loft drier (Shanghai, China).

540

Coating of the inclusion bodies with Fe3O4 nanomaterials. The 10 mg Fe3O4 were

541

suspended in 9 mL 100 mM buffers with different pH values (100 mM citrate-sodium

542

citrate buffer for pH 5.5, 100 mM phosphate buffer for pH 6.0-8.0, 50 mM tris-HCl

543

buffer for pH 9.0 and 100 mM borax-sodium hydroxide buffer for pH 9.5). Then 1 mL

544

enzyme solution with 20 mg inclusion bodies was added into Fe3O4 solution and

545

stirred for less than 10 min at 4 °C. The supernatant was first separated from the solid

546

materials by placing a neodymium disk magnet (25 mm diameter). The protein

547

content of the supernatant was measured using the Bradford assay. The amount of

548

immobilized TD was therefore calculated by subtracting this figure from the blank.

549

The solid was eventually washed 3 times with phosphate buffer (pH 7.5) with the help

550

of neodymium disk magnet for separation. Thus, the catalytic activity of the supported

25


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 47

551

enzyme should not be affected by any unimmobolized enzyme, which might possess a

552

high activity. The coating of the inclusion bodies with Fe3O4 nanomaterials was

553

confirmed by infrared spectroscopy.

554

The scanning electron microscope (SEM) pictures were taken in a Hitachi

555

S-4800 fieldemission scanning electron microscope (Hitachi, Japan) in a secondary

556

electron (SE) mode. Samples were prepared by drying on top of a silver substrate a

557

droplet of a suspension of particles dispersed in ethanol with a thin (ca. 20 nm) carbon

558

coating. The transmission electron microscopy (TEM) pictures were taken in JEOL

559

JEM 2010F field emission gun microscope (JEOL, Japan) operating at an acceleration

560

voltage of 200 kV. Samples were prepared by drying carbon-coated copper grid a

561

droplet of a suspension of particles dispersed in ethanol.

562

Infrared spectroscopy. IR spectra were measured with a Nicolet Nexus 670

563

spectrometer (Nicolet, USA) equipped with a mercury cadmium telluride (MCT)

564

detector, and purged with dry nitrogen. For ATR-FTIR measurement, all samples were

565

scanned in an out-of-compartment horizontal ATR accessory with a high-throughput

566

73×10×6 mm, 45o trapezoidal germanium crystal (IRE). The purified WT enzyme was

567

separated from the solution using ultrafiltration with molecular weigh cut-off

568

(MWCO) 30-50 kDa, and then was washed twice using D2O. The purified WT

569

enzyme was finally suspended in D2O for ATR-FTIR measurement. The inclusion

570

bodies ∆11, ∆25 and ∆35 were centrifuged for 5 min at 12,000 × g, and then was

571

washed twice using D2O. Final inclusion bodies were suspended in D2O for

572

ATR-FTIR measurement. The final protein spectra were used for further analysis. All

26


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ACS Catalysis

573

illustrated spectra were shown after band narrowing by Fourier self-deconvolution

574

(FSD) to resolve overlapping IR bands at FSD parameters γ=2.2 and f=3.0, where γ is

575

the band narrowing factor and f is a high-pass filter.27-28 Subtraction and FSD

576

calculation were performed by using OMNIC software (Nicolet, USA). The curve

577

fitting of IR spectra was performed with Origin 8.0 (Origin lab, USA). IR spectra of

578

∆25, Fe3O4 and Fe3O4/∆25 were measured using powder. ∆25 and Fe3O4/∆25 were

579

freeze-dried by using Biosafer-10D lyophilizer (Biosafer, China).

580

Yield of 2-OBA before and after immobilization. Biosynthesis of 2-OBA was

581

assayed in the reaction mixtures under different conditions. Reactions were carried

582

out at 30 oC and 200 rpm on a reciprocating shaker. The supernatant of immobilized

583

enzymes was separated using neodymium disk magnet for 2 min while the reaction

584

mixture of TD inclusion bodies was centrifuged at 10,000 × g for 10 min, and the

585

concentration of 2-OBA in the supernatant was analyzed. Optimum conditions were

586

also conducted. For transformation in different pH, the experiments were carried out

587

for 8 h in different buffer (50 mM phosphate buffer for pH 6.0-8.0, and 50 mM

588

tris-HCl buffer for pH 9.0) with 3.4±0.1 µM enzyme and 1 M L-Thr at 30 oC. During

589

the transformation, the solution was kept at their initial pH with 20% ammonia and

590

the temperature was kept at 30 oC. For transformation in different temperature, the

591

experiments were carried out for 8 h in 50 mM phosphate buffer (pH 7.0) with

592

3.4±0.1 µM enzyme and 1 M L-Thr. During the transformation, the pH was kept 7.0

593

with 20% ammonia and the temperature was kept from 30 to 60 oC. These

594

experiments were all conducted using thermostatic water bath oscillators (Boxun,

27


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

595

Page 28 of 47

Shanghai, China) with 160 rpm.

596

Recycle capacity for TD inclusion bodies and immobilized TD inclusion bodies

597

was also conducted. Transformation experiments were carried out for each 1 h in 50

598

mM phosphate buffer (pH 7.0) with 3.4±0.1 µM enzyme and 0.1 M L-Thr. During the

599

transformation, the solution was kept at pH 7.0 with 20% ammonia and the

600

temperature was kept at 30 oC. The supernatant of immobilized enzymes was

601

separated using neodymium disk magnet for 2 min while the reaction mixture of TD

602

inclusion bodies was centrifuged at 10,000 × g for 5 min, then they were used again.

603

The immobilized protein after recycling for 20 times was determined by SDS-PAGE

604

analysis (12% acrylamide). All these experiments were carried out for three times and

605

error bars showed the standard deviation.

606

Structure modeling and MD simulations. The structural models of TD truncated

607

variants were acquired by homology modeling using SWISS-MODEL workspace

608

(http://swissmodel.expasy.org/), while the WT enzyme structure (PDB: 1TDJ) was

609

downloaded from PDB database (http://www.rcsb.org/). Structure analysis of enzymes

610

was conducted using Pymol software.80 The hydrophobicity plot of wild type TD

611

enzyme was achieved by summiting their primary sequence to website

612

(https://web.expasy.org/protscale/).23, 81 A hydrophobicity score (Roseman algorithm,

613

window = 9) higher than zero denotes increased hydrophobicity. All molecular

614

dynamics simulations were performed using GROMACS version 5.0.2 with the

615

AMBER99SB-ILDN force field.82-83 Each system was immersed in an octahedron

616

box of TIP3P water molecules, which extended 12 Å from the dissolved atoms in all

28


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ACS Catalysis

617

three dimensions. Na+ or Cl− counterions were added to neutralize the systems. The

618

protonation state of residues was set according to pH 7.0. And then steps including

619

energy minimization, system equilibration, and production protocols were carried out

620

according to the literatures.84-85 Following steepest-descent energy minimization,

621

2-nanosecond NVT simulations were run at 298 K in 2-femtosecond steps. Finally 30

622

ns simulations were run at 298 K in 2 fs steps.

623 624

Supporting information

625

The Supporting Information is available free of charge on the ACS Publications

626

website.

627

SDS-PAGE analysis of WT and truncated threonine deaminase, ITC of TD variant

628

solution with Ile added, radius of gyration (Rg) of WT and truncated threonine

629

deaminase, characteristics of Fe3O4 nanomaterials, effect of the ion strength, glycerin,

630

PEG4000 and F127 on the IB activities and adsorption using Fe3O4, effect of metal

631

ions on wild type TD, SDS-PAGE after recycling for 20 times, primers used for

632

plasmid construction, kinetic constants for TDs activities of the wild-type and

633

truncated enzymes.

634 635

Notes

636

The authors declare no competing financial interest.

637 638

Acknowledgment 29


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 47

639

This work was supported by National First-class Discipline Program of Light

640

Industry Technology and Engineering (LITE2018-06), National High-tech R&D

641

Program of China (863 Program, No.2015AA021004), Jiangsu Province Science

642

Fund for Distinguished Young Scholars (BK20150002), National Natural Science

643

Foundation of China (31570085 and 21778024), China Postdoctoral Science

644

Foundation Funded Project (2017M620189), the 111Project (111-2-06), Fundamental

645

Research Funds for the Central Universities (JUSRP51708A), and the Priority

646

Academic Program Development of Jiangsu Higher Education Institution and the

647

Jiangsu province "Collaborative Innovation Center for Advanced Industrial

648

Fermentation" industry development program.

30


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649

References

650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691

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859

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Page 36 of 47

860 861

Figure 1. Model structures of WT enzyme and its truncation variants ∆11, ∆25 and

862

∆35. (A) An overall view of TD tertiary structure (PDB: 1TDJ). In WT structure, s15,

863

s17, s16 and s12 formed a tight structure with the help of C terminal α-helix h18,

864

while these β-sheets was exposed in the solvent in the structure of truncation variants

865

(the hydrophobic residues were marked in brown). The structure of truncation

866

peptides was marked in different color (red for ∆35, yellow for ∆25 and green for

867

∆11). (B) RMSF calculated from MD simulations for the wild type TD at room

868

temperature. The residues chosen for designing inclusion bodies by truncation was 36


Page 37 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

869

highlighted in orange and the residue range was shown at the right top. (C)

870

Hydrophobicity plot of TD as a function of their primary sequence. A hydrophobicity

871

score (Roseman algorithm, window = 9) higher than zero denotes increased

872

hydrophobicity. The β-sheets exposed to solution after C-terminal truncation were

873

highlighted in blue and the residue range was shown at the bottom. (D) Model of

874

inclusion body formation for truncation variants. Hydrophobic interaction occurred

875

among the truncated C-terminal regulatory domain and resulted in the dimer

876

aggregation with the formation of TD inclusion bodies. Surface with enzymatic

877

activities was colored in yellow and the inner side of inclusion bodies was colored in

878

blue with 60% transparency. (E) SEM and TEM images of inclusion bodies (Bars

879

marked inside the pictures).

37


ACS Catalysis

NE 0.5V 0.3I 0.3I+3V

25 0

0

25

50

75

100

40 30

∆11

-0.5

20

∆25

-2.0

0

-2.5

0

25

50

75

Thr concentration (mM)

25

50

75

0

100

10

100

20

30

40

50

Time (min)

∆35

-30

30

-1

∆H (kJ mol )

10

0

40

-1 -1

20

0

-1.5


Activity (µmol mg min )

-1 -1

30

-1.0

10


40

0.0

Dp (µW)

50

-1

75

WT

-1

100


-1

B

-1


A


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20

10

-40 -50 -60 -70

0

25

50

75


100

0

2

4

6

8

Molar ratio

38


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ACS Catalysis

880

Figure 2. Study of effectors on the WT enzyme and its truncation variants. (A) Steady-state kinetics of WT enzyme and its truncation variants

881

∆11, ∆25 and ∆35. Assays were carried out without addition of effectors (NE, ■) or in the presence of 0.5 mM Val (○ ○), 0.3 mM Ile (△ △) or 0.3

882

mM Ile plus 3 mM Val (▽ ▽). Ile concentrations used in these experiments were close to the IC50 values determined in Figure 3. All the data were

883

fitted with the Hill equation. (B) Isothermal titration calorimetry of WT enzyme solution with Ile added. Upside indicated the raw data-heat

884

plotted against injection sequence when 5 µM WT enzyme solution is exposed to increasing amounts of Ile (concentration of 200 µM) as

885

described in the text. Downside indicated the integrated enthalpy change after subtraction of heat of dilution plotted against mole ratio of Ile to

886

WT enzyme.

887

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40


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ACS Catalysis

889

Figure 3. Effects of Ile and MD simulations of the WT enzyme and its truncation variants ∆11, ∆25 and ∆35. (A) The inhibition of enzymatic

890

activity by Ile for WT enzyme and its truncation variants ∆11, ∆25 and ∆35. Reactions were monitored in buffer containing 50 mM threonine.

891

The data corresponding to WT were fitted with a Hill equation [IC0.5 (inhibition) =0.34±0.03 mM and nH=1.09±0.13]. (B) Yield of 2-OBA using

892

WT enzyme and its truncation variants ∆11, ∆25 and ∆35 in either the presence or absence of 2 mM Ile. The p value between truncation variants

893

and the wild type were all

Relieving Allosteric Inhibition by Designing Active Inclusion Bodies

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