Thermostability and specific activity enhancement of an arginine

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,. 6. Jiangsu 214122, China. 7. ‡. International Joint Laboratory on F...
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Functional Structure/Activity Relationships

Thermostability and specific activity enhancement of an arginine deiminase from Enterococcus faecalis SK23.001 via semi-rational design for L-citrulline production Xue Cai, Hangyu Jiang, Tao Zhang, Bo Jiang, Wanmeng Mu, and Ming Miao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02858 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 29, 2018

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Thermostability and specific activity enhancement of an arginine deiminase from

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Enterococcus faecalis SK23.001 via semi-rational design for L-citrulline

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production

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† † *,† †,‡ †,‡ Xue Cai , Hangyu Jiang , Tao Zhang , Bo Jiang , Wanmeng Mu , Ming

5

Miao

6 7 8 9





State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

Jiangsu 214122, China ‡

International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu

214122, China

10 11

*

Corresponding author.

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Address: State Key Laboratory of Food Science and Technology, Jiangnan University,

13

Wuxi, Jiangsu 214122, P. R. China.

14

Tel.: (86) 510-85919161. Fax: (86) 510-85919161.

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E-mail: [email protected] (T.Zhang).

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ABSTRACT L-Citrulline

is a non-essential amino acid with a variety of physiological functions

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and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The

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enzymatic production approach is of immense interest because of the mild condition,

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high yield, low cost and environmental benignity. However, the major hindrances of

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L-citrulline

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Hence, in this work, directed evolution and site-directed mutagenesis aided with in

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silico screening, including b-factor values and HoTMuSiC, were applied to an ADI

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from Enterococcus faecalis SK23.001 (EfADI) identified previously and a triple-site

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variant R15K/F269Y/G292P was obtained. The triple-site variant displays a 2.5-fold

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higher specific enzyme activity (333 U mg-1), lower Km value of 6.4 mM and a

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6.1-fold longer half-life time (t1/2, 45 °C = 86.7 min) than the wild-type EfADI. This

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work provides a protein engineering strategy to improve the enzyme activity and

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thermostability, which might be transferrable to ADIs and other enzymes.

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KEYWORDS:

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thermostability, directed evolution, site-directed mutagenesis

industrialization are the poor thermostability and enzyme activity of ADI.

L-citrulline,

arginine

deiminase,

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Enterococcus

faecalis,

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INTRODUCTION

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L-citrulline

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functions and wide applications in the health care, pharmaceutical and fine chemical

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industries.1 In health care, L-citrulline is able to protect DNA and polymorphonuclear

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leukocytes (PMNs) from oxidative damage,2 to maintain the normal function of the

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cardiovascular system,3 to reduce gut injury during exercise and to stimulate muscle

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protein synthesis4.

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is a naturally occurring non-essential amino acid with various biological

Previously, three methods for

L-citrulline

production have been reported,

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including chemical synthesis, microbial fermentation and enzymatic synthesis.5-7 The

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enzymatic synthesis is of most interest because of its mild reaction conditions, high

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production yield, and simple purification of final products.6 This approach involves

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one enzyme, arginine deiminase (ADI, EC 3.5.3.6), which hydrolyzes L-arginine into

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L-citrulline

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microorganisms have been characterized, including Aspergillus fumigatus (A.

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fumigatus),8 Bacillus cereus (B. cereus),9 Enterococcus faecalis (E. faecalis),10

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Euglena gracilis (E. gracilis),11 Halobacterium salinarium (H. salinarium),12

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Lactococcus lactis (L. lactis),13 Mycoplasma arginine (M. arginine),14 Pseudomonas

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putida (P. putida),15 Pseudomonas plecoglossicida (P. plecoglossicida),16 and

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Streptococcus pyogenes (S. pyogenes).17 The recombinant ADIs with the highest

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specific enzyme activity were reported from L. lactis, Lactococcus lactis ssp. Lactis

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(L. lactis ssp. lactis), and E. faecalis with values of 195.7 U mg-1, 140.3 U mg-1, and

and ammonium. Currently, a number of ADIs from various

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131.2 U mg-1, respectively. 6,10,13 The most stable ADI was from L. lactis ssp. lactis

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with the half-life time of 90 min at 50 °C.13 The L-citrulline production by ADI is

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commercially limited due to the low enzyme activity and thermostability of the

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characterized ADIs under processing conditions. To increase L-citrulline production

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commercially, natural ADIs need to be engineered to enhance their catalytic behaviors,

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including enzyme activity, thermostability and kinetic parameters.

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Protein engineering can be divided into three main categories: irrational design,

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semi-rational design and rational design.18 The combination of irrational and rational

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design constitutes the semi-rational design, which is an effective approach that intends

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to generate small but functionally rich mutant libraries.19-22 Therefore, we employ an

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combinatorial approach (directed evolution and site-directed mutagenesis) on our

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previously constructed recombinant ADI from E. faecalis SK23.001 (EfADI)

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expressed in Escherichia coli (E. coli) BL21(DE3)10, which displayed a specific

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enzyme activity of 131.2 U mg-1 on L-arginine at pH 5.5 and retained 50% of its initial

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activity after 14.3 min at 45 °C, with the intention of achieving thermostability

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improved variants. In this study, directed evolution via error-prone polymerase chain

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reaction (epPCR), in silico screening (B-factor values and the HoTMuSiC algorithm)

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and site-directed mutagenesis were successively applied to the wild-type EfADI,

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resulting in variants with enhanced enzyme activities and thermostabilities.

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MATERIALS AND METHODS

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Bacterial Strains, Plasmids, and Materials. The recombinant plasmid

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pET28a(+)-EfADI was constructed in our previous study.10 E. coli DH5α and

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BL21(DE3) cells were used as hosts for cloning and overexpression of EfADI,

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respectively. The vector pET28a(+) (Novagen, Darmstadt, Germany) was used for

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construction of the recombinant vector harboring mutant genes. E. coli cells were

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incubated at 37 °C in Luria-Bertani media (peptone 10 g L-1, yeast extract 5 g L-1,

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NaCl 10 g L-1) containing kanamycin (50 µg mL-1). Instant Error-Prone PCR Kit was

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purchased from Tiandz, Inc. (Beijing, China). Plasmid extraction kit, agarose gel

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DNA recovery kit, DNA purification kit, isopropyl β-D-1-thiogalactopyranoside

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(IPTG), kanamycin and reagents of analytical grade were purchased from Sangon

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Biotech (Shanghai, China). Restriction enzymes BamH I and Xho I, DNA ligase,

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DNA marker and premixed protein marker (Broad) were purchased from Takara

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Biomedical Technology Inc. (Shiga, Japan). The nucleotide sequence of E. faecalis

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SK23.001 ADI is available in the GenBank database (accession number: MG763888).

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Mutant Library Generation by Error-Prone PCR. The gene of EfADI was

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subjected to epPCR to introduce random mutations following the Instant Error-Prone

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PCR Kit manual. EfADI gene (1.26 kb) was amplified in a reaction volume of 30 µL

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that contained 10 ng of the ancestral pET28a(+)-EfADI plasmid, 10× epPCR mix, 2.5

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units of Taq polymerase, 10 pM primer ADI_forward with BamH I site (underlined)

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(5′-CGCGGATCCATGAGTCATCCAATTAATGT-3′) and 10 pM primer ADI_reverse

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with Xho I site (underlined) (5′-CCGCTCGAGTTAAAGATCTTCACGGT-3′), 10×

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dNTP specific for epPCR, 3 µL of 5 mM MnCl2, and double distilled water. The

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reaction program was as follows: 94 °C for 3 min, 30 cycles of 94 °C for 60 s, 45 °C

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for 60 s, 94 °C for 60 s. The resulting PCR products were purified using DNA

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purification kit and subsequently digested with BamH I and Xho I at 37 °C for 1 h.

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The PCR products were then cloned into pET28a(+) vector and transformed into E.

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coli BL21(DE3).

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Expression and Screening of EfADI Variants. Colonies from the mutant library

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constructed by epPCR were screened for improved enzyme activity following Ni’s

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high-throughput screening strategy22 with minor modifications. Briefly, single

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colonies were inoculated into 5 mL of LB medium with kanamycin (50 µg mL-1) and

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IPTG (0.5 mM) and cultivated at 37 °C for 24 h. The cells were collected by

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centrifugation (8, 000 × g, 4 °C, 10 min) and resuspended in 200 µL of reaction buffer

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(10 g L-1 L-arginine, 20 mM potassium phosphate buffer, pH 6.0). The reaction

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solutions were incubated at 45 °C for 15 min, followed by addition of 200 µL of 10%

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sulfuric acid to terminate the reaction. After adding 60 µL of diacetyl monoxime

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thiosemicarbazide solution, reaction tubes were incubated at 37 °C for 2 h. Active

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colonies resulted in the formation of purple color with a λmax of 530 nm.

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Homology Modeling, In silico Screening and MD Simulation. Multiple amino

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acid sequence alignments was performed using the online server Clustal Omega23 and

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ESPript 3.024. Three-dimensional homology models of the wild-type EfADI and five

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variants

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R15K/F269Y/G292P) were generated by SWISS-MODEL25 using S. pyogenes ADI

(M1(R15K),

R15K/G292P,

R15K/G264P,

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R15K/F269Y,

and

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(PDB entry: 4BOF) as template and visualized by PyMOL 2.0.6 (DeLano Scientific

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LLC, San Carlos, CA, USA). Molecular docking was performed using Autodock 4.2

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with a genetic algorithm to investigate the molecular interactions.26

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In silico screening, including B-FITTER and HoTMuSiC algorithm, was carried

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out to select the amino acids for substitutions. B-FITTER is applied in the quest to

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enhance the thermal robustness of EfADI. This program calculates the average

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b-factor value of all the atoms around an amino acid in a given protein, excluding

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hydrogen.27 The input was a .pdb file, and the output was a ranking of amino acid

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residues according to b-factor values. HoTMuSiC algorithm is performed to evaluate

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the changes in melting temperature.28

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To further evaluate the thermostability of the wild-type EfADI and the triple-site

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variant (R15K/F269Y/G292P), molecular dynamics (MD) simulation was performed

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with Gromacs 5.1 package29 at 450 K for 50 ns. Structures were put in a cubic box

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and solvated and systems were minimized in energy and equilibrated in temperature

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and pressure using the Berendsen coupling algorithm and the Rahman-Parrinello

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algorithm, respectively. All bonds were constrained using the LINear Constraint

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Solver (LINCS) algorithm, and periodic boundary conditions were applied. The cutoff

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value for van der Waals interactions was set at 1 nm, and electrostatic interactions

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were calculated using a particle mesh Ewald algorithm. To investigate the stability of

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the proteins, the root-mean-square deviation (RMSD) analyses were performed using

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the available Gromacs analysis tools.

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Site-Directed Mutagenesis. Site-directed mutagenesis of EfADI at positions 264,

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269 and 292 was accomplished using a one-step PCR method with primers listed in

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Table S1. The plasmid pET28a(+)-EfADI-M1 obtained from directed evolution was

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used as template. The PCR system (50 µL) was constituted of pfu Turbo DNA

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polymerase (2.5 U), dNTP mix (10 mM), template (10 ng) and double distilled water.

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The PCR program was as follows: 98 °C for 1 min, one cycle; 98 °C for 30 s, 61 °C

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for 30 s, 72 °C for 7 min, 3 cycles; 98 °C for 30 s, 61 °C for 30 s, 72 °C for 5 min, 15

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cycles; 72 °C, 5 min, one cycle. After Dpn I digestion and purification, the amplified

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PCR products were cloned into pET28a(+) vector, and transformed into E. coli

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BL21(DE3) cells. The sequences of constructed variant genes were verified by DNA

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sequencing.

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Expression and Purification of EfADI Variants from Site-directed

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Mutagenesis. E. coli BL21(DE3) cells harboring pET28a(+)-EfADI-M1 variants

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were cultivated, and disrupted and purified following the methods of the wild-type

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pET28a(+)-EfADI described by Jiang et al.10 The subunit molecular weight of EfADI

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variants were examined using 12% of sodium dodecyl sulfate polyacrylamide gel

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electrophoresis (SDS-PAGE).

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Standard Enzyme Activity Assay. Enzyme activities of M1 (R15K) and its

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variants were assayed based on the amount of produced L-citrulline . Briefly, 10 µL of

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purified protein was incubated with 990 µL of potassium phosphate buffer (20 mM,

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pH 6.0) containing 10 g L-1 L-arginine at 45 °C for 10 min. The reaction was

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terminated in boiling water for 10 min. One unit of enzyme activity was defined as

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the amount of ADI needed to produce 1 µmol of L-citrulline per minute.

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Product Quantitation via HPLC. The contents of L-citrulline and L-arginine in

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the enzymatic reaction mixtures were analyzed following the method as described by

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Jiang et al10, using high-performance liquid chromatography (HPLC) (Series 1200;

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Agilent Technologies, Palo Alto, CA, USA) coupled with a HYPERSIL ODS C18

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column (4.6 × 250 mm, 5 µm,Thermo Fisher Scientific, Waltham, MA, USA), and a

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UV detector (model LC-9A, Shimadzu, Kyoto, Japan). o-Phthaldialdehyde was used

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as precolumn derivation reagent and products were monitored at 338 nm with

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excitation at 262 nm. The column was eluted at 40 °C with a flow rate of 1 mL min-1.

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The gradient mobile phase was composed of buffer A (6.5 g L−1 CH3COONa·3H2O

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including 0.16 mg L−1 triethylamine and 4.4 mg L−1 tetrahydrofuran, pH 7.2) and

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buffer B (24 g L−1 CH3COONa·3H2O, pH 7.2/acetonitrile/methanol (1:2:2 by

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volume)). The A/B ratios were 92:8, 62:38, 0:100, 0:100 and 92:8 at run times of 0,

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20, 24, 25.5 and 28.5 min, respectively.

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Biochemical Characterizations of M1 Variants. The buffer systems,

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HAc-NaAc (20 mM, pH 4.0-6.0), potassium phosphate (20 mM, pH 6.0-7.5), and

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Tris-HCl (20 mM, pH 7.5-9.0) were used for determining the variants’ optimal pHs.

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The highest enzyme activity was assumed to be 100%. The pH stability of enzymes

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was assayed by measuring the residual activities in their respective optimal buffers at

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45 °C for 10 min after incubation in buffers with pH ranges from 4.0 to 9.0 at 4 °C for

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12 h. The enzyme activity before pre-incubation at 4 °C was assumed to be 100%.

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The optimal temperature for each variant was determined over the temperature

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range 10-70 °C. The variant thermostability at 45 °C was also analyzed. Namely, the

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purified enzymes were incubated at 45 °C up to 90 min and samples were taken every

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10 min. Sodium azide (0.1% (w/v)) was added to prevent microbial growth. The

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initial activity level before pre-incubation was defined as 100%. Additionally, the

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thermostability of triple-site variant at 60 °C was measured as above.

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For determination of kinetic parameters, L-arginine concentrations ranged from 1

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to 30 mM were applied and reactions were carried out at 45 °C in their respective

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optimal buffer. The kinetic parameters, Km and Vmax, were calculated by non-linear

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regression fitting using Enzyme Kinetics Module (SigmaPlot 12.5; Systat Software

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GmbH, Erkrath, Germany).

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L-Citrulline

Production with Purified Variants. L-Citrulline was produced in

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45 °C water bath with stirring at 150 rpm for 10 h with 100 g L−1 L-arginine, 10 U of

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purified M1 and its variants at their respective optimal buffer and pH in a total

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volume of 100 mL.

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Statistical Analysis. Statistical analyses and calculation were performed using

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Excel 2011 (Microsoft Corporation, Redmond, WA, USA). All enzymatic assays and

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analytic measurements were performed at least in duplicate.

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RESTULTS AND DISCUSSION

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Screening of Mutant Library. A total number of ~500 EfADI variants after

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epPCR were screened in a parallel manner for increased enzyme activity. One variant

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(M1) with a slightly improved enzyme activity (1.2 fold to the activity of the

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wild-type EfADI) was identified and the sequencing result revealed an Arg15Lys

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mutation. The optimal temperature and pH of variant M1 remained the same as that of

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the wild-type enzyme (55 °C and pH 5.5). The half-life value of variant M1

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(t1/2,M1,45 °C = 14.5 min) was similar to that of the wild-type EfADI (t1/2,WT, 45 °C = 14.3

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min). The sequence alignment result shown in Figure 1 revealed that the mutated

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residue at position 15 of EfADI shares the same residue lysine position in three

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reported ADIs from S. pyogenes17, M. penetrans30 and L. lactis13, indicating that the

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residue lysine might be more preferred here. Moreover, modeled protein structure of

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M1 showed that R15K is on the back of the substrate-binding pocket, which is far

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away from the active site (Figure 2b). Though the enzyme activity value of M1 was

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slightly increased,the thermostability of M1 was not enhanced, which was of the

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most interest in our study. The mechanism behind the activity-improvement of M1

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will be investigated further by site-saturation mutagenesis. No further mutagenesis

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was implemented on position 15 in this study. Instead, in silico screening approaches

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including B-FITTER and HoTMuSiC algorithm were employed on variant M1 for the

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following site-directed mutagenesis with the intention to reduce the mutant library

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and obtain thermostability-enhanced enzymes.

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In silico Screening. The stability of enzymes at high temperature, generally over

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40 °C, are often the prerequisite to be considered in the biotechnological production

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for the prevention of microbial contamination. Therefore, the rational design of

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EfADI engineering based on b-factor values and HoTMuSiC algorithm was carried

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out

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thermostability-improved variants.

using

variant

M1

as

parent

to

provide

us

with

predictions

of

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A three-dimensional structure of M1 was firstly modeled based on the X-ray

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crystal structure of the S. pyogenes ADI (PDB ID: 4BOF, SpADI) using

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SWISS-MODEL. The amino acid sequence identity between M1 and SpADI was

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66.83%, which is higher than the identities of M1 and other reported ADIs, for

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instance, P. aeruginosa (32.75%), M. arginine (38.38%), M. penetrans (39.95%), P.

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plecoglossicida (31.34%) and L. lactis ssp. lactis (47.89%). The amino acid sequence

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alignment revealed that SpADI and M1 share the identical active site (Figure 1). Five

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key amino acids that constituted the active site, correspondingly positioned in M1 are

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Asp162, Glu216, His271, Asp273 and Cys398.30-32 A number of 10 amino acid

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residues with the highest b-factor values calculated by B-FITTER program were listed

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in Table 1 and presented in the modeled structure of M1 (Figure 2c). Figure 2d

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represents the space-filling of M1 structure, where the locations of these ten residues

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are clearly shown on the surface of M1, which may have direct relation to the rise in

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the optimum temperature. Previous studies have shown that the rigidity of surface

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residues is of great importance in the maintenance of protein thermostability.28,30-31

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Therefore, it is a common strategy to modify the surface amino acids for enhanced

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enzyme thermostability.19,20 In addition, abundant researches of protein engineering

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have revealed that the amino acids around the active site are crucial to the enzyme

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activity besides the catalytic triads.33, 34 To achieve a thermostability improved variant

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with minimum time and cost, three residues, Gly292, Phe269 and Gly264, were

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chosen as sites for point mutagenesis because (i) Gly292 possesses the highest

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b-factor value indicating its flexibility in the structure and (ii) Phe269 and Gly264 are

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close to the active site and located on the extruding loop that might be of

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conformational importance for substrate binding (Figure 3).

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Next, instead of site-saturation mutagenesis, site-directed mutagenesis was

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applied on three selected residues for the courtesy of time, labor and expenses. Amino

251

acids substituted were expected to be more rigid than the original amino acids with

252

respect to the changes of the melting temperature calculated by HoTMuSiC, the value

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of which is an indicator of protein structure stability.28 Three selected residues (G292,

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F269 and G264) were in silico mutated and the predicted melting temperature

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changes were summarized in Table S2. As a result, three mutations of each selected

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residue with the highest melting temperature (G292D, G292C, G292P, F269D, F269Y,

257

F269P, G264S, G264F, G264P) were constructed, and their crude enzyme activities at

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45 °C and pH 6.0 were determined to preclude those variants with decreased enzyme

259

activities compared to M1.

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Crude Enzyme Activities of M1 Variants. The crude enzyme activities of M1

261

(R15K) variants at position 292, 269 and 264 (G292D, G292C, G292P, F269D,

262

F269Y, F269P, G264S, G264F, G264P) were determined using L-arginine as substrate

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in 20 mM potassium phosphate buffer, at pH 6.0 and 45 °C for 10 min and the results

264

were summarized in Table 2. Variant R15K/G292P displayed an enzyme activity

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value of 16.65 U mL-1, and variants R15K/G264P and R15K/F269Y showed enzyme

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activity values (6.77 and 6.42 U mL-1) similar to that of M1 (parent, 6.66 U mL-1).

267

The enzyme activities of other variants decreased to 7-33% of M1. Therefore, three

268

variants, R15K/G292P, R15K/F269Y and R15K/G264P, out of nine M1 variants, were

269

selected for the enzyme characterizations including pH and temperature profiles and

270

kinetic parameters.

271

Effects of pH and Temperature on M1 and Its Variants. The pH profile and

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pH stability of variant M1 (R15K), R15K/G292P, R15K/F269Y and R15K/G264P

273

were investigated and shown in Figure 4a and 4b, respectively. Variant M1 (R15K),

274

used as parent of site-directed mutagenesis, displayed an optimal pH of 5.5 that was

275

the same as that of the wild-type EfADI10. All three variants, R15K/G292P,

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R15K/F269Y and R15K/G264P, showed an optimal pH shift in favor of neutral

277

environment from 5.5 to 6.5, 6.0 and 6.5, respectively (Figure 4a). In particular,

278

variant R15K/G264P exhibited over 90% of its enzyme activity in a broad pH range

279

from 5.5 to 7.5 (Figure 4a) and retained over 80% of its enzyme activity after 12 h

280

incubation in a pH range from 6.5 to 7.5 (Figure 4b). The pH range of variant

281

R15K/G292P also expanded (pH 6.0-7.5) with over 80% of its enzyme activity

282

retained. The optimal pH of an enzyme is influenced by various factors, such as the

283

pKa of the active site residues, enzyme-substrate binding, or enzyme surface charge.38

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In case of EfADI, residues G292P, F269Y and G264P were located on the surface of

285

the protein and was speculated that the surface charges were changed due to the

286

mutation. However, further experiments (e.g. ionization status and pKa value of each

287

residue) should be carried out, to gain an insight into the mechanism of pH shift by

288

mutations on residues G292P, F269Y and G264P.

289

M1 variants, including R15K/G292P, R15K/F269Y and R15K/G264P, exhibited

290

an identical molecular weight of 49 kDa in SDS-PAGE (Figure S1) and optimal

291

temperature of 55 °C as that of the wild-type EfADI and variant M1 (Figure 4c). The

292

thermostabilities of M1 and three variants were evaluated at 45 °C (Figure 4d), at

293

which temperature the bioconversion of L-arginine was carried out by EfADI10. The

294

half-lives (t1/2, 45°C) of the wild-type EfADI, M1 and its variants were listed in Table 3.

295

The half-life of variant R15K/G264P was similar to that of the wild-type EfADI and

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M1. The results showed a remarkable increase in half-life values of variant

297

R15K/F269Y (t1/2, 45 °C = 48.0 min) and R15K/G292P (t1/2, 45 °C = 82.5 min), which

298

were ~3.3 fold and ~5.7 fold to the half-life value of the wild-type EfADI (t1/2, 45 °C =

299

14.3 min), respectively. Interestingly, the P. plecoglossicida ADI variant M13-9

300

harbored a residue substitution at position 276 (Ala to Trp) which is in consistent with

301

the mutated position of EfADI at residue 269 (Figure 1).16 The enhanced

302

thermostability of ADI in both cases indicated that the residue at position 269 plays

303

an important role in protein stabilization. As variant R15K/G292P showed a

304

remarkable improvement in thermostability, it implies that the residue on position 292

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305

is crucial to the enzyme stability as well.

306

Specific Activities and Kinetics of M1 and Its Variants. The specific activities

307

and kinetics of M1 and its variants (R15K/G292P, R15K/F269Y and R15K/G264P)

308

were determined and tabulated in Table 3. Variant R15K/G264P and F269Y exhibited

309

similar specific activity as the wild-type EfADI and their parent enzyme M1.

310

Noticeably, R15K/G292P displayed a significantly enhanced specific activity value of

311

296.8 U mg-1 at pH 6.5, which is ~2.5-fold of the wild-type EfADI. Variants

312

R15K/G292P and R15K/F269Y both exhibited decreased Km values to 6.4 and 9.3

313

mM in comparison with that of the wild-type EfADI (Km = 10.1 mM). As expected, an

314

increased catalytic efficiency (Kcat/Km = 55.4 s-1 mM-1) was obtained for R15K/G292P

315

compared with the values of the wild-type EfADI (Kcat/Km = 30.6 s-1 mM-1) and M1

316

(Kcat/Km = 23.1 s-1 mM-1). Because variant R15K/G292P resulted in an increased

317

specific activity and catalytic efficiency, a lower Km and an extended half-life time

318

discussed above, meanwhile variant R15K/F269Y exhibited an increased catalytic

319

efficiency and half-life time, therefore, a triple-site variant (R15K/F269Y/G292P) will

320

be constructed for the investigation of its enzymatic properties.

321

Properties of A Triple-site EfADI Variant (R15K/F269Y/G292P). Recent

322

studies have shown that a double-site or even triple-site variant exhibited superior

323

properties compared with a single-site variant.19, 20 Therefore, based on the results of

324

enzyme activity, pH stability, thermostability and kinetics of the double-site variants

325

(R15K/G292P, R15K/F269Y, R15K/G264P), a triple-site variant R15K/F269Y/G292P

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of EfADI was constructed to achieve an enzyme with improved enzyme activity and

327

thermostability.

328

After cultivation and purification, the enzymatic properties of the triple-site

329

variant R15K/F269Y/G292P were studied. It showed an optimal pH and temperature

330

of 7.5 and 55 °C (Figure 4a and 4c), with a specific activity of 332.8 U mg-1. The

331

specific activity was increased by 11% compared to the double-site variant

332

R15K/G292P (296.8 U mg-1). The triple-site variant displayed the lowest substrate

333

affinity towards arginine of 6.4 mM and the highest catalytic efficiency of 57.0 s-1

334

mM-1

335

R15K/F269Y/G292P increased further to 86.7 min at 45 °C. Its thermal stability at

336

60 °C was tested and the half-life time was 12 min. The previous studies have

337

reported some ADIs with good thermal stability as well. For instance, an ADI from L.

338

lactis ssp. lactis ATCC 7962 was presented with the half-lives of 90 min at 50 °C and

339

15 min at 60 °C16 and a P. plecoglossicida ADI variant (M13-9) showed an increased

340

half-life value of 17.5 min at 60 °C (parent: 4 min at 60 °C)16. Interestingly, the

341

optimal pH value of the variant R15K/F269Y/G292P showed a 2-unit shift to pH 7.5

342

compared to wild-type EfADI. Thus, this triple-site mutation at position 15, 269 and

343

292, in some aspects, offers the possibility of site-directed mutagenesis to those ADIs

344

studied for anti-tumor drugs, e.g. P. plecoglossicida ADI36, which should work in the

345

physiological environment (pH 7.4), but showed lower enzyme activity comparing to

346

that of its natural enzyme.

among

all

the

EfADI

variants.

The

half-life

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of

variant

Journal of Agricultural and Food Chemistry

347

Additionally, we performed MD simulations at 450 K for 50 ns for variant M1

348

(parent) and variant R15K/F269Y/G292P using the modeled structures for a deeper

349

understanding of its conformational changes. As shown in Figure 5, the backbone

350

root-mean-square deviation (RMSD) values were similar in the first 30 ns, however,

351

variant R15K/F269Y/G292P showed a lower RMSD value (maximum 0.79 nm)

352

during the last 20 ns whereas variant M1 displayed an increment after 44 ns to an

353

RMSD value of 1.28 nm, suggesting that variant R15K/F269Y/G292P was more

354

stable than M1 during the simulations, which is in consistence with the experimental

355

results of half-life values.

356

Bioconversion of L-Citrulline. The bioproductions of L-citrulline by wild-type

357

EfADI and its variants were shown in Figure 6. All the reactions can finally reach the

358

conversion rate of 95.8%, 96.5%, 97.5%, 96.0%, 93.2% and 97.4% at 45 °C,

359

corresponding to wild-type EfADI, M1, R15K/G264P, R15K/G292P, R15K/F269Y

360

and R15K/G292P/F269Y in 4 h, 5 h, 4 h, 4 h, 10 h and 5 h, respectively. In particular,

361

variant R15K/G264P reached 95% conversion rate after 2 h incubation, almost half

362

the time needed for the wild-type to reach the same conversion rate. The conversion

363

rate of variant R15K/G292P/F269Y (97.4%) is higher than that of wild-type (95.8%).

364

Song et al. reported an ADI from Lactococcus lactis of 92.6% conversion rate after 8

365

h incubation at 50 °C6 (Table 4). In summary, the results demonstrate the

366

improvement of L-citrulline production by EfADI variants and high productivity of

367

L-citrulline

can be achieved by enzymatic conversion, which provides preliminary

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basis for industrial scale production of L-citrulline. Structural

Analysis

of

EfADI

and

the

Triple-site

Variant

370

R15K/F269Y/G292P. To gain deeper insights of the rationale behind the effects of

371

mutations at position 292 and 269 on the activity and thermostability enhancement,

372

the modeled structures of EfADI and its variant R15K/F269Y/G292P with substrate

373

L-arginine

374

ID: 1RXX), group A. streptococcus (PDB ID: 4BOF), Mycoplasma penetrans (4E4J)

375

and M. arginini (PDB ID: 1LXY) have been experimentally analyzed.17,

376

Comparative crystallographic studies have shown that the entrance to the active site is

377

decorated with four loops (loop 1: 37-44 aa, loop 2: 174-178 aa, loop 3: 263-271 aa,

378

loop 4: 393-400 aa) that undergo conformational transitions upon arginine binding

379

(Figure 3).39 In variant R15K/F269Y/G292P, one of the mutated amino acids, Phe269

380

was located on loop 3. The substitution tyrosine was similar to the previous amino

381

acid phenylalanine in structure with one more hydroxide group on the benzene ring.

382

However, the interactions of Phe or Tyr with the surrounding amino acids were

383

different and were shown in Figure 7a and 7d, respectively. Phenylalanine (F269) was

384

found one hydrogen bond with H266, whereas the mutated residue tyrosine (F269Y)

385

showed three additional hydrogen bonds including His287, Glu289 and Glu242.

386

Furthermore, F269Y was covalently bonded with M270, the latter of which composed

387

a covalent bond with H271 that was one of the five key amino acids in the active site.

388

The mutated residue proline at position 292 showed interactions with E291 and G293,

were discussed. By far, crystal structures of ADI from P. aeruginosa (PDB

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and further with E289 and F269Y, whereas G292 presented no interactions with the

390

surrounding amino acids (Figure 7b and 7e). Therefore, the reinforced interactions

391

between G292P or F269Y with the surrounding amino acids, especially on the protein

392

surface, might (i) improve the rigidity of the structure due to an increased number of

393

hydrogen bonds and covalent bonds, (ii) increase the steric hindrance of other cavities

394

near the active site and thus facilitate the correct entrance of the substrate into the

395

active site, and (iii) expediate the conformational changes in the loop 3 and hence

396

promote the arginine conversion (Figure 7c and 7f). Consequently, the improved

397

thermostability and enzyme activity of the triple-site variant R15K/F269Y/G292P of

398

an arginine deiminase from E. faecalis SK23.001 offers a possibility in anti-tumor

399

drugs or a large-scale L-citrulline production. In addition, the structural analysis

400

revealed an increased number of hydrogen and covalent bonds between the mutated

401

residues (G292P and F269Y) and the surrounded residues that might explain for its

402

increased stability and enzyme activity.

403 404

ASSOCIATED CONTENT

405

AUTHOR INFORMATION

406

Corresponding Author

407

* E-mail: [email protected]. Phone: (86) 510-85919161.

408

ORCID

409

Bo Jiang: 0000-0002-0638-6456

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Wanmeng Mu: 0000-0001-6597-527X ACKNOWLEDGMENTS

412

Many thanks go to Dr. Hinawi A. M. Hassanin (Jiangnan University) for his help

413

regarding the structure modeling and the docking of EfADI and L-arginine and M.S.

414

Qiuming Chen (Jiangnan University) for MD simulation analysis.

415

CONFLICT OF INTEREST

416 417 418 419

The authors declare no competing financial interest. COMPLIANCE WITH ETHICAL STANDARDS This article does not contain any studies with human participants or animals performed by any of the authors.

420

Supporting Information. Changes of melting temperature with amino acid

421

substitutions at position G292, F269 and G264 calculated by the online server

422

HoTMuSiC (Table S1), primers for site-directed mutagenesis (Table S2) and

423

SDS-PAGE analysis (Figure S1).

424 425

REFERENCES

426

(1) Kaore, S.; Amane, H.; Kaore, N. Citrulline: pharmacological perspectives and its

427

role as an emerging biomarker in future. Fundam. Clin. Pharmacol. 2013, 27, 35-50.

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(2) Sureda, A.; Cordova, A.; Ferrer, M. D.; Tauler, P.; Perez, G.; Tur, J. A.; Pons, A.

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Effects of

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oxidative burst and nitric oxide production after exercise. Free Radic. Res. 2009, 43,

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oral supplementation on polymorphonuclear neutrophils

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828-835.

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(3) Figueroa, A.; Alvarez-Alvarado, S.; Jaime, S. J.; Kalfon, R.

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supplementation attenuates blood pressure, wave reflection and arterial stiffness

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responses to metaboreflex and cold stress in overweight men. Br. J. Nutr. 2016, 116,

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279-285.

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(4) Bourdon, A.; Parnet, P.; Nowak, C.; Tran, N. T.; Winer, N.; Darmaun, D.

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L-citrulline

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intrauterine growth restriction. J. Nutr. 2016, 146, 532-541.

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(5) Hao, N.; Mu, J.; Hu, N.; Xu, S.; Yan, M.; Li, Y.; Guo, K.; Xu, L. Improvement of

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L-citrulline

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J. Ind. Microbiol. Biotechnol. 2015, 42, 307-313.

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(6) Song, W.; Sun, X.; Chen, X.; Liu, D.; Liu, L. Enzymatic production of L-citrulline

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by hydrolysis of the guanidinium group of L-arginine with recombinant arginine

444

deiminase. J. Biotechnol. 2015, 208, 37-43.

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(7) Fang, F.; Zhang, J.; Zhou, J.; Zhou, Z.; Li, T.; Lu, L.; Zeng, W.; Du, G.; Chen, J.

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Accumulation of citrulline by microbial arginine metabolism during alcoholic

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fermentation of soy sauce. J. Agric. Food Chem. 2018, 66, 2108-2113.

448

(8) El-Sayed, A. S.; Hassan, M. N.; Nada, H. M. Purification, immobilization, and

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biochemical characterization of L-arginine deiminase from thermophilic Aspergillus

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fumigatus KJ434941: anticancer activity in vitro. Biotechnol. Prog. 2015, 31,

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396-405.

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supplementation enhances fetal growth and protein synthesis in rats with

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(9) Li, L.; Li, Z.; Wang, C.; Xu, D.; Mariano, P. S.; Guo, H.; Dunaway-Mariano, D.

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The electrostatic driving force for nucleophilic catalysis in L-arginine deiminase: a

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combined experimental and theoretical study. Biochemistry 2008, 47, 4721-4732.

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(10) Jiang, H.; Huang, K.; Mu, W.; Jiang, B.; Zhang, T. Characterization of a

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recombinant arginine deiminase from Enterococcus faecalis SK32.001 for L-citrulline

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production. Proc. Biochem. 2018, 64, 136-142.

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(11) Park, B.; Hirotani, A.; Nakano, Y.; Kitaoka, S. Purification and some properties of

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arginine deiminase in Euglena gracilis. J. Agric. Food. Chem. 1984, 48, 483-489.

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(12) Monstadt, G. M.; Holldorf, A. W. Arginine deiminase from Halobacterium

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salinarium. Purification and properties. Biochem. J. 1991, 273, 739-745.

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(13) Kim, J. E.; Jeong, D. W.; Lee, H. J. Expression, purification, and characterization

463

of arginine deiminase from Lactococcus lactis ssp. lactis ATCC 7962 in Escherichia

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coli BL21. Protein Expr. Purif. 2007, 53, 9-15.

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(14) Noh, E. J.; Kang, S. W.; Shin, Y. J.; Kim, D. C.; Park, I. S.; Kim, M. Y.; Chun, B.

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G.; Min, B. H. Characterization of mycoplasma arginine deiminase expressed in E.

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coli and its inhibitory regulation of nitric oxide synthesis. Mol. Cells. 2002, 13,

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137-143.

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(15) Su, L.; Ma, Y.; Wu, J. Extracellular expression of natural cytosolic arginine

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deiminase from Pseudomonas putida and its application in the production of

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L-citrulline. Bioresour Technol. 2015, 196, 176-83.

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(16) Ni, Y.; Li, Z.; Sun, Z.; Zheng, P.; Liu, Y.; Zhu, L.; Schwaneberg, U. Expression of

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arginine deiminase from Pseudomonas plecoglossicida CGMCC2039 in Escherichia

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coli and its anti-tumor activity. Curr. Microbiol. 2009, 58, 593-598.

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(17) Henningham, A.; Ericsson, D. J.; Langer, K.; Casey, L. W.; Jovcevski, B.;

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Chhatwal, G. S.; Aquilina, J. A.; Batzloff, M. R.; Kobe, B.; Walker, M. J.

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Structure-informed design of an enzymatically inactive vaccine component for group

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A Streptococcus. MBio. 2013, 4, e00509-e00513.

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(18) Stepankova, V.; Bidmanova, S.; Koudelakova, T.; Prokop, Z.; Chaloupkova, R.;

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Damborsky, J. Strategies for stabilization of enzymes in organic solvents. ACS Catal.

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2017, 3, 2823-2836.

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(19) Zhang, W.; Jia, M.; Yu, S.; Zhang, T.; Zhou, L.; Jiang, B.; Mu, W. Improving the

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thermostability and catalytic efficiency of the

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Clostridium bolteae ATCC BAA-613 using site-directed mutagenesis. J. Agric. Food

485

Chem. 2016, 64, 3386-3393.

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(20) Yu, S.; Zhang, Y.; Zhu, Y.; Zhang, T.; Jiang, B.; Mu, W. Improving the catalytic

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behavior of DFA I-Forming inulin fructotransferase from Streptomyces davawensis

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with site-directed mutagenesis. J. Agric. Food Chem. 2017, 65, 7579-7587.

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(21) Jamil, S.; Liu, M.-H.; Liu, Y.-M.; Han, R.-Z.; Xu, G.-C.; Ni, Y. Hydrophobic

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mutagenesis and semi-rational engineering of arginine deiminase for markedly

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enhanced stability and catalytic efficiency. Appl. Biochem. Biotechnol. 2015, 176,

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1335-1350.

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(22) Ni, Y.; Liu, Y.; Schwaneberg, U.; Zhu, L.; Li, N.; Li, L.; Sun, Z. Rapid evolution

D-Psicose

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of arginine deiminase for improved anti-tumor activity. Appl. Microbiol. Biotechnol.

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2011, 90, 193-201.

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(23) Sievers, F.; Higgins, D. G. Clustal Omega for making accurate alignments of

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many protein sequences. Protein Sci. 2018, 27, 135-145.

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(24) Robert, X.; Gouet, P. Deciphering key features in protein structures with the new

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ENDscript server. Nucleic Acids Res. 2014, 42, W320-W424.

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(25) Guex, N.; Peitsch, M. C.; Schwede, T. Automated comparative protein structure

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modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective.

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Electrophoresis 2009, 30, S162-S173.

503

(26) Wojciechowski, M. Simplified AutoDock force field for hydrated binding sites. J.

504

Mol. Graph. Model 2017, 78, 74-80.

505

(27) Reetz, M. T. Laboratory evolution of stereoselective enzymes: a prolific source of

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catalysts for asymmetric reactions. Angew. Chem. Int. Ed. Engl. 2011, 50, 138-174.

507

(28) Pucci, F.; Bourgeas, R.; Rooman, M. Predicting protein thermal stability changes

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upon point mutations using statistical potentials: Introducing HoTMuSiC. Sci. Rep.

509

2016, 6, 23257.

510

(29) Abraham, M. J.; Murtola, T.; Schulz, R.; Pall, S.; Smith, J. C.; Hess, B.; Lindahl,

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E. GROMACS: High performance molecular simulations through multi-level

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parallelism from laptops to supercomputers. Software X 2015, 1-2, 19-25.

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(30) Gallego, P.; Planell, R.; Benach, J.; Querol, E.; Perez-Pons, J. A.; Reverter, D.

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Structural characterization of the enzymes composing the arginine deiminase pathway

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in Mycoplasma penetrans. PLoS One 2012, 7, e47886.

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(31) Lu, X.; Li, L.; Wu, R.; Feng, X.; Li, Z.; Yang, H.; Wang, C.; Guo, H.; Galkin, A.;

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Herzberg, O.; Mariano, P. S.; Martin, B. M.; Dunaway-Mariano, D. Kinetic analysis

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of Pseudomonas aeruginosa arginine deiminase mutants and alternate substrates

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provides insight into structural determinants of function. Biochemistry 2006, 45,

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1162-1172.

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studies of arginine deiminase catalysis: the protonation state of the Cys nucleophile. J.

523

Phys. Chem. B 2011, 115, 3725-3733.

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(33) Yu, S. H.; Wang, X.; Zhang, T.; Jiang, B.; Mu, W. M. Probing the role of two

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critical residues in inulin fructotransferase (DFA III-Producing) thermostability from

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Arthrobacter sp. 161MFSha2.1. J. Agr. Food Chem. 2016, 64, 6188-6195.

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(34) Zhu, L.; Cheng, F.; Piatkowski, V.; Schwaneberg, U. Protein engineering of the

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antitumor enzyme PpADI for improved thermal resistance. Chembiochem 2014, 15,

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276-283.

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(35) Ding, H.; Liu, H.; Yin, Y.; Ding, Y.; Jia, Y.; Chen, Q.; Zou, G.; Zheng, Z. Insights

531

into the modulation of optimum pH by a single histidine residue in arginine deiminase

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from Pseudomonas aeruginosa. Biol. Chem. 2012, 393, 1013-1024.

533

(36) Zhang, L.; Liu, M.; Jamil, S.; Han, R.; Xu, G.; Ni, Y. PEGylation and

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pharmacological characterization of a potential anti-tumor drug, an engineered

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arginine deiminase originated from Pseudomonas plecoglossicida. Cancer Lett. 2015,

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357, 346-354.

537

(37) Das, K.; Butler, G. H.; Kwiatkowski, V.; Clark, A. D., Jr.; Yadav, P.; Arnold, E.

538

Crystal structures of arginine deiminase with covalent reaction intermediates;

539

implications for catalytic mechanism. Structure 2004, 12, 657-667.

540

(38) Gallego, P.; Planell, R.; Benach, J.; Querol, E.; Perez-Pons, J. A.; Reverter, D.

541

Structural characterization of the enzymes composing the arginine deiminase pathway

542

in Mycoplasma penetrans. PLoS One 2012, 7, e47886.

543

(39) Galkin, A.; Lu, X.; Dunaway-Mariano, D.; Herzberg, O. Crystal structures

544

representing the Michaelis complex and the thiouronium reaction intermediate of

545

Pseudomonas aeruginosa arginine deiminase. J. Biol. Chem. 2005, 280,

546

34080-34087.

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Figure captions

548

Figure 1. Multiple sequence alignment of arginine deiminases enzymes from S.

549

pyogenes (4BOF.A), E. faecalis (EFQ16080.1), P. aeruginosa (1RXX.A),

550

Mycoplasma arginine (1LXY.A), M. penetrans (4E4J.A), P. plecoglossicida

551

(ABS70718.1), L. lactis sub sp. (AGV74030.1). The alignment was prepared with

552

Clustal Omega23 and ESPript 3.024. Five key amino acids in the active site, D162,

553

E216, H271, D273 and C398, are denoted with blue circles. R15, G292, F269 and

554

G264 are denoted with green triangles. Strictly conserved residues are highlighted in

555

red, while equivalent residues are boxed in blue. The secondary structure elements of

556

S. pyogenes ADI are presented above the corresponding sequence. Helices are

557

represented as spirals and β-strands are represented as arrows. Turns are indicated

558

with TT.

559 560

Figure 2. Three-dimensional models. (a) Structure superposition of S. pyogenes

561

(4BOF) ADI (blue) and EfADI (gray). (b) Variant M1 of EfADI with a mutation at

562

position 15 from Arg to Lys. (c) 10 amino acids with high b-factor values in EfADI

563

were shown in sticks and colored in orange. Substrate arginine was docked in the

564

active site via AUTODOCK. (d) Space-filling representation of EfADI monomer of

565

the same angle as (c) showing the 10 amino acids with high b-factor values, which are

566

colored in orange, are located on the surface of the protein.

567

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568

Figure 3. Partial structural model of the wild-type EfADI in the active site. Five key

569

amino acids, D162, E216, H271, D273 and C398 were denoted in red. The residues

570

for site-mutagenesis, F269 and G292, were colored in yellow. Substrate arginine was

571

docked in the active site by AUTODOCK. Loop 1, 2, 3 and 4 were colored in blue.

572

The modeled protein structure was constructed by SWISS-MODEL and visualized

573

using PyMOL 2.0.6 (Schrödinger, LLC).

574 575

Figure 4. pH and temperature profiles of EfADI variants including M1 (R15K,

576

orange, diamond), R15K/G264P (blue, circle), R15K/F269Y (green, triangle),

577

R15K/G292P (purple, square), and R15K/F269Y/G292P (red, cross). (a) pH profiles

578

of EfADI variants. The assays were carried out at 45 °C for 10 min with L-arginine as

579

substrate in pH buffers ranging from 4.0 to 9.0 in 20 mM using HAc-NaAc buffer (pH

580

4.0-6.0), potassium phosphate buffer (pH 6.0-7.5), and Tris-HCl (pH 7.5-9.0). (b) pH

581

stability of EfADI variants. The enzymes were incubated in various pH for 12 h and

582

the residual enzyme activities were measured at pH 6.0 and 45 °C. (c) The optimal

583

temperature of EfADI variants. The enzymes were incubated with L-arginine at

584

temperatures from 30 °C to 70°C in the optimal pH of each variant for 10 min. (d)

585

Thermostabilities of the variants at 45 °C. The enzymes were incubated at 45 °C for

586

up to 90 min. The activity without heat treatment was defined as 100%. Error bars

587

correspond to the standard deviations of three independent determinations.

588

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589

Figure 5. Time dependence of RMSD values of wild-type EfADI (blue) and triple-site

590

variant R15K/F269Y/G292P (red) after simulation at 450 K for 50 ns.

591 592

Figure 6. Enzymatic production of L-citrulline by wild-type EfADI and its variants

593

using L-arginine as substrate. The symbols were: WT (black, inverse triangle), M1

594

(R15K, orange, diamond), R15K/G264P (blue, circle), R15K/F269Y (green, triangle),

595

R15K/G292P (purple, square), and R15K/F269Y/G292P (red, cross). Reaction

596

conditions: 100 g L-1 L-arginine with 10 U purified enzyme and 45 °C for 10 hours at

597

their respective optimal buffer and pH.

598 599

Figure 7. Structural features of EfADI and the triple-site variant R15K/F269Y/G292P

600

in the active site and the mutated residue. (a) and (b) presented the structural models

601

of interactions between residue Phe269 and Gly292 with the surrounding residues in

602

the wild-type EfADI, respectively. Red is oxygen, and blue is nitrogen. (d) and (e)

603

presented the structural models of the interactions between mutated residue

604

Phe269Tyr and Gly292Pro with the surrounding residues in the triple-site variant

605

R15K/F269Y/G292P, respectively. In a, b, d and e, red is oxygen, and blue is nitrogen.

606

(c) and (f) showed the space-filling representation of wild-type EfADI and variant

607

R15K/F269Y/G292P. The loops outside the active site are colored in blue, residues at

608

position 269 and 292 with its interacted residues are colored in green and pink,

609

respectively. The hydrogen bonds were presented with yellow dashed lines. The

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610

figure was generated using PyMOL 2.0.6 (Schrödinger, LLC). Tables Table 1. The 10 amino acid residues with the highest b-factor values of arginine deiminase from E. faecalis SK23.001. Three amino acid residues chosen for subsequent site-directed mutagenesis were highlighted in bold.

No.

Position Amino acid residue B-factor value

1

292

Gly

91.3

2

290

Ile

81.8

3

269

Phe

81.2

4

288

Pro

78.9

5

302

Lys

78.8

6

308

Gln

78.0

7

132

Pro

77.1

8

40

Asp

76.1

9

297

Tyr

75.6

10

264

Gly

75.0

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Table 2. Crude enzyme activities of nine M1 variants (100% vol. EAarginine=6.66 U mL-1).

Variant

Relative EA (%)

Variant

Relative EA (%)

M1

100

F269Y

96.4

G292P

253.6

F269D

21.5

G292D

13.6

G264F

9.3

G292C

9.5

G264P

102.6

F269P

32.9

G264S

6.9

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Table 3. Specific activities, the half-lives and kinetic parameters of the wild-type EfADI and its variants with residue substitutions at positions 292, 269 and 264.

Specific EA

t1/2 at 45°C

Km

Kcat

Kcat / Km

(U mg-1)

(min)

(mM)

(s-1)

(s-1 mM-1)

Wild-type EfADI*

131.2±5.3

14.3

10.1±0.3

309±13

30.6

M1 (R15K)

143.2±3.2

14.5

11.6±0.5

268±11

23.1

R15K/G264P

134.7±3.5

14.9

12.5±0.4

291±12

23.3

R15K/F269Y

126.5±4.3

48.0

9.3±0.2

342±13

36.8

R15K/G292P

296.8±5.1

82.5

6.7±0.4

371±8

55.4

R15K/F269Y/G292P

332.8±2.9

86.7

6.4±0.5

365±16

57.0

Variant

*The data of wild-type EfADI was obtained from our previous report by Jiang et al.10

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Table 4. Comparison of characteristics of recombinant arginine deiminases from various microorganisms. Microorganism Pseudomonas plecoglossicida (PpADI)
 PpADI variant M9 (K5T/D38H/D44E/A128T/ V140L/E296K/F325L/H404R) Pseudomonas putida Lactococcus lactis ssp. lactis Lactococcus lactis Aspergillus fumigatus

Streptococcus pyogenes Mycoplasma arginini Enterococcus faecalis (EfADI)

Opt. pH

Opt. Temp

Specific EA -1

t1/2

Km, L-arginine

Kcat

L-Citrulline

-1

Reference

(−)

(°C)

(U mg )

(min/h/day)

(mM)

(s )

Yield*

6.0

37

4.76

2.9

0.2

NR

16

7.4

37

NR

~5 min (50 °C)a ~4 min (60 °C) 3.5 days (37 °C)

NR

13.7

NR

34

6.0 7.2

37 60

101.2 U mL-1 140.3

NR 8.67

NR NR

100% (3 h) NR

15 13

7.2 5.0-8.0

50 57

195.7 26.7

3.5 8.76

NR 17.2a

92.6% (8 h) NR

6 8

6.5 6.4 5.5

37 41 55

1.1 72.3 131.2

1.33 0.37 10.1

NR NR 309

15 h (37 °C) 90 min (50 °C) 15 min (60 °C) NR 12.1 h (40 °C) 6.9 h (50 °C) 5.6 h (70 °C) NR 5 days (37 °C) 14.3 min (45 °C)

NR 17 NR 14 96.9% 10 (2 h, 45 °C) EfADI triple-site variant 7.5 55 332.8 86.7 min (45 °C) 6.4 365 97.4% This study (R15K/F269Y/G292P) 12 min (60 °C) (5 h, 45 °C) * Otherwise no additional description, the reaction was carried out at the optimal pH and temperature, substrate concentration is 100 g L-1. a Data calculated from the original paper by the author.

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NR, not reported.

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Figure Graphics Figure 1.

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Figure 2.

a

b

c

G292

d

Y297 Q308

R15K

P288 I290 F269

K302 G264 Arginine

D40

P132

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Figure 3.

G292 F269 Loop 4

D273 Loop 3

C398 Arginine Loop 1

H271 E216

D162

Loop 2

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Figure 4. 120

120

100

Relative Enzyme Activity (%)

Relative Enzyme Activity (%)

a 80

60

40

20

b

100

80

60

40

20

0

0 3

4

5

6

7

8

9

10

3

4

5

6

pH (-) 120

8

9

10

120

c 100

Relative Enzyme Activity (%)

Relative Enzyme Activity (%)

7

pH (-)

80

60

40

d

100

80

60

40

20

20

0

0 25

30

35

40

45

50

Temperature

55

60

65

70

75

0

10

20

(oC)

30

40

50

Time (min)

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60

70

80

90

100

Journal of Agricultural and Food Chemistry

Figure 5.

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Figure 6.

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Figure 7.

a

E289

H287

G292

K299

G293

I290

E289

F269

Q346

c

G292

Wild-type EfADI

V275

b

L294 H266

T274

I290 K268

F269

H271 Arginine

I290

H287

G292P

e

G293

E289

V275

G292P

E291 E289

I290

f

E301 L294

Q346 F269Y

K268

H266

F269Y

H271

M270 Arginine

E242

R267

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Variant R15K/F269Y/G292P

d

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TOC Graphic

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