Computation-aided rational deletion of C-terminal region improved the

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Biotechnology and Biological Transformations

Computation-aided rational deletion of C-terminal region improved the stability, activity and expression level of GH2 #-glucuronidase Beijia Han, Yuhui Hou, Tian Jiang, Bo Lv, Lina Zhao, Xudong Feng, and Chun Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03449 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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Journal of Agricultural and Food Chemistry

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Computation-aided rational deletion of C-terminal region improved

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the stability, activity and expression level of GH2 β-glucuronidase

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Beijia Han†,⊥, Yuhui Hou†,⊥, Tian Jiang†, Bo Lv†, Lina Zhao§, Xudong Feng*,†, Chun Li*,†

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†Institute

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and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China.

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§Key

for Synthetic Biosystem/Department of Biochemical Engineering, School of Chemistry

Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High

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Energy Physics, Chinese Academy of Sciences, Beijing 100049, China.

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*Correspondence

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⊥These authors

to Xudong Feng ([email protected]) and Chun Li ([email protected])

contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT

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In this study, computation-aided design on the basis of structural analysis was employed to

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rationally identify a highly dynamic C-terminal region that regulates the stability, expression level

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and activity of a GH2 fungal glucuronidase from Aspergillus oryzae Li-3 (PGUS). Then, four

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mutants with a precisely truncated C-terminal region in different lengths were constructed, among

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them, mutant D591-604 with a 3.8-fold increase in half-life at 65 °C and a 6.8 kJ/mol increase in

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Gibbs free energy showed obviously improved kinetic and thermodynamic stability compared to

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PGUS. Mutants D590-604 and D591-604 both showed approximately 2.4-fold increases in the

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catalytic efficiency kcat/Km and 1.8-fold increases in the expression level. Additionally, the

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expression level of PGUS was doubled through a C-terminal region swap with bacterial GUS from

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E. coli (EGUS). Finally, the robust PGUS mutants D590-604 and D591-604 were applied in the

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preparation of glycyrrhetinic acid with a 4.0- and 4.4-fold increases in concentration through

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glycyrrhizin hydrolysis by a fed-batch process.

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Keywords: β-glucuronidase; molecular dynamics simulation; protein engineering; C-terminal

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region; glycyrrhetinic acid

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INTRODUCTION

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Glucuronide conjugates are an important class of natural product compounds, in which the

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bioactive aglycone is decorated with glucuronic acids. Glucuronides can be derived by

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glucuronidation based on various carbon frameworks such as terpenoids, flavones and alkaloids,

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which were widely applied in the food, cosmetic and pharmaceutical industry.1-3 The glucuronic

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acid moieties play an important role in regulating the solubility, function and bioavailability of the

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glucuronides, but overdecoration with glucuronic acid may also introduce negative effects.4-6

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Traditionally, the excess sugar is removed by acid/base hydrolysis, but this process needs a high

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pressure and high temperature, and the reaction is uncontrollable. A more efficient and green

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process for the transformation of glucuronides is highly desirable. β-Glucuronidases (GUSs, EC

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3.2.1.31), as an important class of glycoside hydrolases (GHs), cleave the β-glucuronidic bond

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from the nonreducing end and release the glucuronic acid moieties.7 Most GUSs identified so far

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belong to GH2, and the crystal structures of GUS from humans (HGUS), Escherichia coli (EGUS),

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and Firmicutes in GH2 have been resolved.8-10 Recently, GUSs have drawn special attention in

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modifying natural glucuronides due to the mild reaction conditions and controllable process.5, 6, 11,

46

12

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the complexed components in the natural products may also inhibit the activity of GUSs. Therefore,

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increasing the stability and activity of GUSs under harsh reaction conditions has become an

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important issue, as it will assist the enzyme in resisting the deactivation of the intricate substrate

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and allow elevating the reaction temperature to increase the reaction efficiency.13, 14

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However, the poor solubility of the natural products has limited the hydrolysis efficiency, and

Directed evolution is the most widely used way to improve enzyme stability, but this method 3



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is labor-intensive and requires high-throughput screening.15-18 With the development of structural

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biology, many relationships between the enzyme structure and the stability/other functions have

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been revealed. The mutagenesis of a single or several key residues based on structural analysis or

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sequence alignment has been reported to improve the enzyme stability, which significantly

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simplifies the experimental task.19-22 Recently, loop engineering has drawn special attention to

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improving the enzyme stability due to the exposure to the solvent and the likeliness of establishing

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interactions with the substrate.23, 24 Flexible surface loops have been considered “hot regions” for

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engineering, therefore, loop truncation

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significantly improve the enzyme stability. However, it is more important to precisely target these

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loops for mutagenesis due to the associated large reorganization of the tertiary structure. Although

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several strategies have been proposed based on the B-factor or sequence alignment, more efforts

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are still required to achieve rational design for identifying unstable loops. Recently, computational

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modeling methods such as molecular dynamics (MD) simulations have also been applied to assist

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in mutation design,30-33 providing ideas and a theoretical basis to guide experiments. Therefore, it

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is anticipated that the combination of structural analysis and computation may be a promising

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pathway for the efficient design of mutants.

25-27

and replacement

28, 29

have been reported to

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Glycyrrhizin (GL), the main compound in licorice, has anti-inflammatory, anti-allergic, and

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anti-cancer activities, but its strong polarity decreases its bioavailability.34-37 GL can be

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transformed into glycyrrhetinic acid (GA) by removing two glucuronic acid moieties. GA has

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better absorption efficiency and all the functions of GL, therefore, was considered an important

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pharmaceutical ingredient.37, 38 In our group, a β-glucuronidase was identified from Aspergillus 4


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oryzae Li-3 and heterologously expressed in Escherichia coli (PGUS), where it hydrolyzed GL

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into GA with high efficiency.4 However, the poor stability and low activity of PGUS greatly

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increased the reaction cost, severely limiting its industrial application. Recently, we resolved the

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crystal structure of PGUS (PDB ID: 5C70), which represents the first structure of a fungal GUS.4

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In this research, the stability, expression level and activity of PGUS were simultaneously improved

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by rationally deleting its C-terminal region, which was a surface dynamic region identified by

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structural analysis and MD simulation, that regulated the function of PGUS. The C-terminal region

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deletion of PGUS enhanced not only enzyme stability but also the expression level and catalytic

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efficiency. By investigating the functional roles of the C-terminal regions of other GH2 GUSs, we

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found that this region was an evolutionary feature of GH2 GUSs. Our work not only presents a

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robust industrial biocatalyst for the modification of natural glucuronide compounds but also

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illustrates the implication of the C-terminal region in GH2 GUSs evolution.

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MATERIALS AND METHODS

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Materials

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The pgus (GenBank, EU095019.1) gene encoding β-glucuronidase from Aspergillus oryzae Li-3

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was saved in our lab.4 The egus gene was directly cloned from E. coli BL21(DE3) strain, and the

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hgus gene was synthesized by Hongxun (Beijing, China) based on the deposited protein sequence

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(GenBank, AAA52561.1). E. coli TOP10 and BL21(DE3) competent cells were purchased from

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Biomed (Beijing, China). The DNA polymerase, restriction enzymes and T4 ligase were purchased

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from TransGene Biotech (Beijing, China). GL and 4-nitrophenyl-β-D-glucuronide (pNPG) were 5



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purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium glycyrrhizinate was purchased

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from Xinjiang Tianshan Pharmaceutical Co. (China). All other chemicals were of analytical grade.

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Bioinformatic analysis

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The values of the B-factor and root mean square fluctuation (RMSF) were used to evaluate the

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protein flexibility, and the high value represented high flexibility. The B-factor was calculated

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with B-FITTER39 software from the PGUS crystal structure, and the RMSF was calculated with

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VMD (Visual Molecular Dynamics) software from the MD simulation.40 The loops with high B-

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factors and RMSF values were considered unstable regions of PGUS. The PGUS mutants with

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deleted unstable loops were computationally constructed with VMD software, and the structures

102

of PGUS and mutants were displayed and analyzed by PyMOL2.7.

103

MD simulation

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In the crystal structure of dimeric PGUS (PDB ID: 5C70), several regions were missed due to the

105

poor electron density. Therefore, these missed regions were first reconstructed by molecular

106

modeling. The missed residues of loop 25-29, loop 364-372 and loop 593-604 were added to the

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PGUS structure by using the built-in psfgen package of VMD software combined with the

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top_all27_prot_lipid (the topology file). Then, the conformation optimization of the reconstructed

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enzyme was facilitated by relaxing only the added amino acids until the complete system became

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stable. Based on this structure, mutants with deleted C-terminal regions were generated by

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removing relevant amino acids, and the missed H or OH at the end of the broken peptide bond was

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added. The obtained structure was used as starting point for the all-atom MD simulation.

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Each system was solvated using TIP3P41 with the water box extended to 15 Å. Periodic 6


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boundary conditions were applied for energy minimization and equilibration, and the solvated

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protein was ionized using 0.15 M NaCl to neutralize the charge. All MD simulations were

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performed with NAMD 2.9. The initial speed of all atoms was set to zero, and then, their relative

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locations were changed instantly to identify the lowest energy spot, which was used as the MD

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simulation original state. After minimization, the temperature was increased from 0 K to 310 K in

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the NVT, and long-range electrostatic effects were modeled using the partial-mesh Ewald

120

method.42 The switching function was employed to guarantee that the van der Waals and

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electrostatic forces could transit to zero smoothly at the cutoff point. All bonds with hydrogen

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atoms were fixed to decrease the computation task. The trajectory was recorded every 5000 steps

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(1 step being 2 fs), and the par_all27_prot_lipid parameter was applied to all calculations in the

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CHARMM force field.

125

Construction of GUS mutants

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The three different GUS genes (pgus, egus and hgus) were added into pET-28a (+) independently

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and used as the template for mutagenesis. Briefly, the forward primer containing the EcoR I site

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and the reverse primer (matching the desired mutants) containing the Not I site were designed and

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used to amplify the GUS genes (the primers are listed in Table S1). The scheme of the plasmid

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construction is shown in Fig. S1. The PCR products were ligated by T4 DNA ligase and

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transformed to E. coli TOP10 competent cells to enrich the plasmid. The resulting plasmids were

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transferred to E. coli BL21(DE3) competent cells for protein expression. The gene sequencing was

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performed by Genewiz (Beijing, China).

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Enzyme expression and purification

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The seed solution of GUSs wild-type and mutants were incubated overnight in Luria-Bertani

136

medium containing 50 μg/mL kanamycin sulfate at 37 °C with shaking. Then, the seed was

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inoculated into a fresh Luria-Bertani medium at a 1% volume fraction and cultured under the same

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conditions. When OD600 reached approximately 0.5-0.6, the protein expression was induced by

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adding 1 mM isopropyl β-D-thiogalactoside (IPTG), followed by incubation at 16 °C for 14 h. The

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cells were collected by centrifugation at 6000 rpm for 10 min and then resuspended in buffer A

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(50mM Tris-HCl, pH 7.4, 150 mM NaCl). Then, the cells were lysed by a high-pressure crushing

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apparatus at 4 °C, and after centrifugation at 12000 rpm for 20 min, the supernatant was reserved

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as a crude enzyme. The target enzyme was eluted with 15% buffer B (50 mM Tris-HCl, pH 7.4, 1

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M imidazole) and desalted with a PD-10 desalting column with 50 mM Tris-HCl (pH 7.3) (GE

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Healthcare, Beijing, China) as an elution buffer. All enzyme purification was conducted in an

146

ÄKTA purifier (GE Healthcare, Beijing, China), and the enzyme molecular weight, expression

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level and purity were analyzed by SDS-PAGE. The protein concentration was assayed at 595 nm

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by the Bradford method according to the standard of bovine serum albumin.43

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Enzyme activity assay and kinetics characterization

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The activity of PGUS, EGUS and the mutants was determined by pNPG hydrolysis. The reaction

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was initiated by adding 20 μL purified enzyme into 30 μL 0.79 mM pNPG (pH 5, 50 mM acetate

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buffer), followed by incubation at 40 °C for 5 min. The reaction was halted by adding 150 μL 0.4

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M NaCO3, and the OD405 of the resulting sample was measured by ELx808 (BioTek, Beijing,

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China) to evaluate the produced pNP. One enzyme unit was defined as the amount of enzyme 8


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required to produce 1 μmol 4-nitrophenol in one minute under the above reaction conditions. The

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Michaelis−Menten kinetics were also evaluated by pNPG hydrolysis, with the concentration

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ranging from 0.12 to 2.76 mM. The kinetic parameters were calculated by nonlinear fitting with

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the curve fitting program of MATLAB.

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Stability determination

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The kinetic thermostability of PGUS, EGUS and the mutants was determined by incubation at

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70 °C for 100 min. Samples were taken every 20 min and put in an ice bath immediately for 20

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min to recover the reverse deactivation. The residual activity was calculated by the pNPG assay as

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described above. The activity of the unincubated sample was taken as 100%.

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The thermodynamic stability of PGUS and the mutants was determined by guanidine

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hydrochloride (GdnHCl)-induced denaturation. The purified enzyme (0.2 mg/mL) was incubated

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in 0.12 M-4.4 M GdnHCl solution containing 10 mM NaCl and 10 mM CaCl2, and the mixture

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was incubated at 25 °C for 2 h. The intrinsic fluorescence of the denaturation process was

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monitored at an excitation wavelength of 280 nm in a 300-mm quartz cell using the fluorescence

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spectrophotometer FluoroMax-4 (HORIBA, USA), and the fluorescence intensity at an emission

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wavelength of 330 nm was recorded for further calculation. ΔG, the Gibbs free energy of unfolding

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in water, was calculated as described by Nick Pace et al.44, 45 In addition, the fluorescence spectrum

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of each enzyme without the addition of GdnHCl was scanned to determine the tertiary structure

173

changes caused by mutation.

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Circular dichroism (CD) analysis

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The CD spectra was measured with a J-810 CD spectropolarimeter (JASCO, Tokyo, Japan) to 9



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investigate the effect of mutation on the enzyme secondary structure. All PGUS enzyme samples

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(0.2-0.3 mg/mL) were prepared in 20 mM NaAC-HAC buffer (pH 4.5) and placed into a quartz

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cell with a 0.1-cm slit. The CD spectra were recorded with far-ultraviolet wavelengths ranging

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from 190 nm to 240 nm, and the CD fitting curve and the content distribution of secondary

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structure were analyzed by jwexpl 32 software.

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Biotransformation of glycyrrhizin

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PGUS, D590-604 and D591-604 were selected for a further GL hydrolysis assay to investigate

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their performance in practical applications. The reactants, consisting of 30 mL crude enzymes and

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70 mL GL (2 g/L ammonium glycyrrhizinate, dissolved in 50 mM acetate buffer, pH 4.5), were

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incubated at 40 °C, and fresh GL was fed to the reactant vessels within a certain interval. Samples

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(97 μL) were taken at certain time and mixed with 3 μL NaOH to stop the reaction, and the

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substrate and product were analyzed by HPLC. The reactant (100 μL) was mixed with 900 μL

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methanol, and the mixture (10 μL) was injected into a C18 column (4.6 × 250 mm, 5-μm particle

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size, Shimadzu). The separation was achieved with a mobile phase consisting of methanol and 6 ‰

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acetic acid solution (84:16 v/v), and the eluate was monitored with an UV detector at 254 nm. The

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GL conversion (CGL) was calculated by CGL(%)=(S0-St)/St×100, where S0 stands for the total GL

192

concentrations provided and St stands for the GL concentration at time t. The GA yield (YGA) was

193

calculated by YGA(%)=SGA/(SGA+SGAMG) × 100, where SGA and SGAMG stand for the molar

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concentrations of GA and glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG), respectively.

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RESULTS

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Discovery of dynamic region by structural analysis and MD simulation 10


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The B-factor is a critical factor to evaluate the flexibility of an individual residue in a protein.46

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However, in our case, the B-factors of several regions of PGUS including loop 25-29, loop 364-

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372 and loop 593-604 were not available owing to the poor electron density,4 therefore these

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missed regions were first reconstructed by MD simulation. RMSF reflects the stability of each

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residue over the MD simulation process. Therefore, a combination of B-factor and RMSF analysis

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was used to target the unstable regions of PGUS. As shown in Fig. 1, RMSF generally corresponds

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well to the B-factor value. Notably, the available part of the C-terminal region from the crystal

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structure showed remarkably high B-factor values with N591 of 83.33 and L592 of 90.01, while

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the rest of the reconstructed C-terminal region also showed a high RMSF, with average value

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above 12 Å. This value is much higher than that of other regions, indicating that the C-terminal

207

region is highly flexible, which may be responsible for the instability of PGUS. The reconstructed

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conformation of the C-terminal region is shown in Fig. 1c. This region formed part of the catalytic

209

TIM-barrel domain but was located far from the substrate channel and active center of PGUS. In

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addition, this region was in a random coil conformation and had no interaction with neighboring

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residues. Theoretically, the deletion of the C-terminal region would not have negative effect on

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the catalytic activity. In contrast, the N-terminal region (residues 1-9) of PGUS was quite stable,

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which was significantly different from the case in enzymes in other GH families, including

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glucanase in GH547 and xylanase in GH11 and GH10,28, 48, 49 where the N-terminal region has been

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reported to play critical roles in enzyme stability. Therefore, on the basis of the above rational

216

analysis, the deletion of the C-terminal region with various lengths was performed to enhance the

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PGUS stability, yielding four mutants: D590-604, D591-604, D595-604 and D597-604 (the 11



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mutants were named DX-X, where X-X stands for the deleted residues). In addition, to compare

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with other GHs, a PGUS mutant with the deleted N-terminal region was also constructed: D1-9.

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To reduce the wet-experiment task, the above five mutants were first constructed by

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computation modeling to test whether the mutagenesis could enhance the enzyme stability via a

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95-ns MD simulation. As shown in Fig. 2a, mutants D590-604 and D591-604 had lower root mean

223

square deviation (RMSD) values than PGUS, indicating that a more stable structure was obtained.

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Mutant D597-604 showed a lower RMSD than the wild-type before 75 ns, but the values then

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became similar. Mutant D595-604 showed a similar RMSD value to PGUS over the entire MD

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simulation. As expected, mutant D1-9 showed an even higher RMSD value than PGUS, indicating

227

that the deletion of the N-terminal region has a detrimental effect on the enzyme stability (Fig. 2b).

228

The positive mutants with a truncated C-terminal region were subjected to experimental tests.

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Deletion of the C-terminal region improved the robustness of PGUS

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PGUS and its mutants were experimentally constructed and purified to a purity higher than 90%

231

(Fig. S2), and their kinetic thermostability was investigated at 65 °C and 70 °C. As shown in Fig.

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3a, mutants D590-604 and D591-604 showed dramatically improved thermostability at 65 °C

233

compared to PGUS. The half-life (t1/2) of D591-604 was estimated to be 693 min based on first-

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order deactivation kinetics, which is 511 min longer than that of PGUS (182 min). D597-604

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showed similar thermostability to PGUS, while D595-604 showed lower thermostability. D590-

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604 and D591-604 still displayed much better performance even at 70 °C, for example, D590-604

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and D591-604 still retained 80% activity after 100 min incubation, while PGUS had only 16%

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activity left. The t1/2 of D591-604 (117 min) was 70 min longer than that of PGUS (47 min) at 12


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70 °C. Incubation for 100 min at 70 °C seemed to be the turning point, since mutants D590-604

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and D591-604 showed severe activity losses after 100 min, with the residual activity decreasing

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from 80% to 34% and 45% in 20 min, respectively. D597-604 showed slightly better

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thermostability than PGUS at the first stage, but it was quickly deactivated after passing the turning

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point. It seems that the thermostability improved with the increasing number of residues deleted

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from the C-terminal region. The thermostability trend is in agreement with the RMSD results in

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Fig. 3, indicating that the computation is reliable for the rational design of mutants.

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The thermodynamic stability of PGUS and the mutants were investigated by monitoring the

247

intrinsic fluorescence variation during guanidine hydrochloride (GdnHCl)-induced denaturation.

248

As shown in Fig. 4, the Gibbs free energies of unfolding (ΔG) of D590-604 and D591-604 were

249

3.0 kJ/mol and 6.8 kJ/mol higher than that of PGUS (31.9 kJ/mol), respectively, indicating that

250

both of the mutants had more stable structures. D595-604 showed a similar ΔG as PGUS (31.6

251

kJ/mol), while D597-604 showed a 10.3 kJ/mol lower ΔG. In summary, mutants D590-604 and

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D591-604, with the deletion of the whole C-terminal region, showed dramatically improved

253

kinetic and thermodynamic stability compared to the wild-type enzyme.

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The kinetics of PGUS and the mutants were characterized by pNPG hydrolysis. As shown in

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Table 1, the Km values of D591-604 and D595-604 were similar to that of PGUS, while the Km

256

values of D590-604 and D597-604 decreased by 58% and 50%, respectively, indicating that these

257

mutants had enhanced binding affinity. In addition, mutant D590-604 showed a similar kcat to

258

PGUS, while all other mutants showed 19-163% increases. In summary, all mutants showed 65-

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143% increases in the catalytic efficiency kcat/Km over that of PGUS. These results indicate that 13



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mutants D590-604 and D591-604 exhibit not only improved stability but also increased catalytic

261

efficiency kcat/Km (increasing by 2.4-fold).

262

The effect of the deletion of PGUS C-terminal region on the enzyme expression level was

263

further investigated. PGUS and mutants D590-604, D591-604 were mostly expressed in soluble

264

form with few inclusion bodies upon comparing the protein contents of the supernatant and whole

265

cell (Fig. 5a). In addition, the expression levels of D590-604 and D591-604 were increased by 1.8-

266

fold compared to that of PGUS, which may be due to that the C-terminal region deletion prompts

267

the correct folding of PGUS, making it more easily expressed. We also investigated the growth

268

curve of three mutants (Fig. S3) and the growth rates of mutants D590-604 and D591-604 were

269

slower than that of wild-type cells, because the heavier expression burden of enzyme repressed the

270

cell growth. Notably, the C-terminal region deletion of PGUS was also beneficial for the enzyme

271

activity. The specific activities of purified mutants D590-604 and D591-604 were 1.4-fold and

272

2.7-fold higher than that of PGUS, respectively, which led the total activity to be 2.5-fold and 4.8-

273

fold higher than that of PGUS (Fig. 5b). Finally, we verified by native PAGE that the C-terminal

274

region deletion did not affect the oligomeric form of PGUS (Fig. S4).

275

The far-UV CD spectra of four mutants were similar to that of PGUS wide-type (Fig. S5).

276

The content distributions of α-helix and β-sheet did not show dramatic changes after the mutation,

277

as the β-sheet continued to be the main form with a content greater than 60% (Table S2). These

278

results indicated that the C-terminal region deletion had no significant effect on the secondary

279

structure. Fluorescence spectra were also analyzed to investigate the effect of deletion of C-

280

terminal region on the tertiary structure of PGUS (Fig. S6). PGUS and four mutants showed a 14


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maximum absorbance at 331 nm, indicating that the mutation had no obvious effect on the tertiary

282

structure of the protein.

283

Investigating the regulating function of the C-terminal region of other GH2 GUSs

284

To investigate whether the C-terminal region has a regulating function for other GH2 enzymes,

285

we also deleted the C-terminal region of a bacterial GUS from E. coli (EGUS), generating two

286

mutants with different C-terminal lengths: D596-603 and D588-603 (the mutants were named by

287

the convention DX-X, where X-X demarcates the deleted residues). As shown in Fig. 6, EGUS

288

showed a tremendously higher thermostability than PGUS, with only a 3% activity loss after 100

289

min incubation at 70 °C. The C-terminal region deletion at various lengths had no clear effect on

290

the EGUS thermostability, since the mutants retained similar activity after 100 min heat-induced

291

denaturation, indicating that the C-terminal region performed no critical role in maintaining

292

stability. Nonetheless, the deletion of the partial C-terminal region (D597-603) increased the

293

soluble expression level by 1.6-fold without any loss of specific activity. Astonishingly, the

294

deletion of the whole C-terminal region of EGUS (D588-603) resulted in a 39.8% decrease in the

295

total expression level and a 49% decrease in the specific activity compared with PGUS, indicating

296

that the partial C-terminal region (residues 588-596) is necessary for maintaining the expression

297

level and activity of EGUS. To further verify its function, we introduced motif 588-596 of EGUS

298

to replace the whole C-terminal region of PGUS and generate a mutant named PGUSE (Fig. S7).

299

As expected, the soluble expression of PGUSE was double that of PGUS, indicating that motif

300

588-596 of EGUS prompts the correct folding not only of EGUS but also of PGUS. Nonetheless,

301

the thermostability of PGUSE was only slightly improved compared with that of PGUS. Therefore, 15



302

it can be concluded that the partial C-terminal region of EGUS (motif 588-596) was a functional

303

region regulating the protein expression. We also tried to heterologously express β-glucuronidase

304

from humans (HGUS) in E. coli, but the protein was mainly expressed in the form of inclusion

305

bodies. Nonetheless, a previous study has shown that HGUS is a lysosomal enzyme, and the whole

306

C-terminal region acts as a retention signal for phosphorylation and targeting the lysosome. When

307

the C-terminal region was deleted, 50% of the activity was lost, and 32-34% less phosphorylated

308

and targeting lysosome was observed.8, 50

309

Application of PGUS mutants in the biotransformation of glycyrrhizin

310

GL is a natural substrate for PGUS. GA, an important pharmaceutical ingredient, was produced

311

by hydrolyzing two glucuronic acid groups from the natural substrate GL via a two-step reaction

312

by PGUS (Fig. 7), and showed better activity and bioavailability. We established a fed-batch

313

process for preparing GA by GL hydrolysis mediated by PGUS and mutants D590-604 and D591-

314

604. As shown in Fig. 8, PGUS hydrolyzed GL in the initial 20 min, with a GL conversion of 66%,

315

and then, the reaction was paused which might be due to the substrate or product inhibition. This

316

point needs further investigation. There was no more GA formed at the final concentration of 0.39

317

g/L, even if more GL was added to the reaction system at 100 min. The GA yield was only 57.1%,

318

indicating that 42.9% byproduct GAMG was also formed due to that the PGUS activity was too

319

low to hydrolyze GAMG into GA. Under the same reaction conditions, for mutants D590-604 and

320

D591-604, GL was rapidly converted in 20 min, with the conversion reaching 88.1% and 84.7%,

321

respectively. The GA yield was 91.9% and 97.4%, respectively, indicating that 8.1% and 2.6%

322

GAMG was formed correspondingly. Then, 0.15 g fresh GL was fed into the reactants at 20 and 16


Page 16 of 39

Page 17 of 39


323

140 min, and the GA concentration continued to increase to 1.71 g/L and 1.58 g/L, respectively,

324

with a GL conversion of 83.6% and 78.1% at 200 min. In addition, the corresponding GA yields

325

were 81.2% and 81.6%, respectively, indicating that GA was the main product. The GL conversion

326

did not continue to increase after the GL feeding at 200 min, which may be due to the enzyme

327

activity was inhibited by the substrate or product. Therefore, two feeding times are optimal for the

328

reaction. All the above results indicate that PGUS mutants D590-604 and D591-604 are more

329

robust catalysts for the industrial application of GL hydrolysis.

330

DISCUSSION

331

Loop engineering such as deletion or swap has been increasingly adopted to manipulate the

332

stability and activity of enzymes. However, the precise targeting of hot-loops for engineering is

333

still quite challenging. In this study, computation-aided design on the basis of structural analysis

334

was employed to rationally identify a regulating C-terminal region that is critical for the low

335

stability of a GH2 glucuronidase PGUS. Then, several mutants with various deleted C-terminal

336

regions were designed that showed not only significantly improved kinetic and thermodynamic

337

stability but also enhanced expression level and activity. All these characteristics are critical for

338

the industrial application of enzymes. This indicates that our proposed method is quite effective in

339

targeting a flexible region for mutation. The deletion of the whole C-terminal region (D590-604,

340

D591-604) conferred better properties on PGUS than partial deletion (D595-604, D597-604),

341

indicating that the C-terminal region is redundant for PGUS to maintain its normal function.

342

Therefore, the C-terminal region deletion may yield a more compact overall structure, resulting in

343

a higher stability (Fig. 3). The thermostability of PGUS mutant D590-604 is higher than that of a 17



344

previously reported thermostable GUS from E. coli obtained by directed evolution, which had a

345

residual activity of approximately 90% after 30 min incubation at 70 °C, while the residual activity

346

of D590-604 was almost 100% under the same conditions.51, 52 In addition, all the PGUS mutants

347

showed similar pH profile as the wild-type (Fig. S8). The C-terminal region deletion was also more

348

favorable for protein folding, resulting in an increased protein expression level. Although the C-

349

terminal region was sterically far from the active site pocket, its deletion yielded an optimal active

350

site pocket conformation that was more favorable for substrate binding (lowering Km) and catalysis

351

(increasing kcat), thus resulting in the increase of the catalytic efficiency. This remote effect of

352

residues to tune catalytic behavior was achieved by conformational changes transmitted

353

throughout the protein backbone, which have also been reported in other enzymes.53-55

354

In addition, it was found that the N-terminal region of PGUS had no obvious impact on the

355

enzyme stability (Fig. 2), which is quite different from the case for enzymes from other GH

356

families such as GH5, GH10 and GH11, where the N-terminal region has been shown to play

357

critical roles in determining the enzyme stability and where a dramatic improvement in the enzyme

358

stability was achieved by deletion25 or exchange with a thermophilic counterpart.49 In addition, the

359

N-terminal coil of the GH10 xylanases was near the C-terminal region to initiate interaction, and

360

engineering this interaction can improve the enzyme thermostability.28, 56 All these results show

361

that the regulating function of the C-terminal region may be a unique feature of GH2 GUSs, which

362

is further evidenced by the fact that the C-terminal region was not conserved from the sequence

363

alignment of typical GUSs in GH2 (Fig. S9). This feature of the C-terminal feature is more

364

dramatic than that of the N-terminal in other reported GH families, since it regulates not only the 18


Page 18 of 39

Page 19 of 39


365

stability, but also the expression level and activity. To support this hypothesis, we also investigated

366

the regulating function of the C-terminal region of a GH2 bacterial GUS from E. coli (EGUS). The

367

deletion of its C-terminal region at various lengths did not result in dramatic improvement in the

368

enzyme stability and activity, which may be because that the stability of EGUS has already well

369

evolved to adapt to high temperature. However, the partial C-terminal region deletion (residues

370

597-603) caused an increased enzyme expression level, and the deletion of the whole C-terminal

371

region (residues 588-603) resulted in a decreased expression level and specific activity, indicating

372

that the partial C-terminal region (residues 588-596) was functional for normal enzyme expression

373

and secretion. Interestingly, we also found this region conferred an increased expression level to

374

PGUS (Fig. S7). HGUS was unsuccessfully expressed in E. coli due to the lack of glycosylation,

375

which is necessary for the proper folding and subunit assembly of HGUS.57 Nevertheless, the C-

376

terminal of HGUS has been demonstrated to have important physiological effects in post-

377

translation, lysosome targeting and maintaining enzyme activity. Therefore, the regulating

378

function of the C-terminal region is dramatically different for GUSs from bacteria, fungi and

379

humans.

380

All these results lead us to conclude that the C-terminal region may be an evolutionary feature

381

of GH2 GUSs. The C-terminal region (14 residues) of PGUS is evolved to be redundant, which

382

suppresses the catalytic capacity, and deleting this redundant part accelerates the evolution process

383

to prompt the folding of GUS into a more robust structure that displays a high expression level,

384

stability and activity. For EGUS, the C-terminal region (15 residues) is partially evolved to be

385

functional, so truncating this region (residues 597-603) improves the expression level with no 19



386

obvious changes in activity and stability, although this impact is not so dramatic for fungal GUS.

387

The functional part of the C-terminal region (residues 588-596) is necessary for the expression and

388

activity of EGUS. Notably, this part can also confer a significantly improved expression level to

389

PGUS (Fig. S7). The C-terminal region of human HGUS is evolved to be fully functional and

390

expanded to contain 20 residues, which are more than those of PGUS and EGUS. A previous study

391

has shown that deleting this part had various negative effects on the enzyme properties such as

392

decreasing the activity and post-translation degree and weakening the lysosome targeting.50 It is

393

anticipated that further examples of this tendency will become apparent in the near future, as more

394

GH2 GUSs from different species are characterized.

395

ASSOCIATED CONTENT

396

Supporting Information

397

The plasmid construction of GUS mutants; The SDS-PAGE, cell growth, native PAGE of PGUS

398

and mutants; The CD and fluorescence spectra of PGUS and mutants; Replacing the C-terminal

399

region of PGUS with partial C-terminal region of EGUS improved its expression level; The C-

400

terminal region of sequence alignment of typical GUSs in the GH2 family; The pH profile of

401

PGUS and mutants; The list of primers; The content of secondary structure.

402

ACKNOWLEDGMENT

403

This research was funded by grants from the National Natural Science Foundation of China (No.

404

21506011, No. 21425624, No. 21878021).

20


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543 544

27



545

List of Figure Legends

546

Figure 1 (a) B-factor and (b) root mean squared fluctuations (RMSF) measured during a 95-ns

547

MD simulation. (c) Ribbon diagram of the conformation of the C-terminal region reconstructed by

548

computational modeling. The C-terminal region is displayed in green, and the adjacent α-helix is

549

shown in orange.

550

Figure 2 Root mean squared deviation (RMSD) of mutants with deleted C-terminal region (a) and

551

N-terminal region (b) measured during a 95-ns MD simulation.

552

Figure 3 Thermostability of PGUS and mutants at (a) 65 °C and (b) 70 °C. Values are averages of

553

three independent replicates; error bars represent average ± one standard deviation.

554

Figure 4 Thermodynamic stability of PGUS and mutants: (a) D590-604, (b) D591-604, (c) D595-

555

604, and (d) D597-604, as determined by monitoring the intrinsic fluorescence of the proteins

556

during their incubation with guanidinium and then fitting these data to the two-state model of

557

protein folding.

558

Figure 5 (a) Expression level of PGUS and mutants D590-604 and D591-604 as analyzed by SDS-

559

PAGE. M: marker; lane 1: PGUS-supernatant; lane 2: PGUS-whole cell; lane 3: D590-604-

560

supernatant; lane 4: D590-604-whole cell; lane 5:D591-604-supernatant; lane 6: D591-604-whole

561

cell; lane 7: No-induction whole cell. The loading volume of the supernatant and whole cell volume

562

was 4 μL. (b) Specific activity of PGUS and mutants characterized by pNPG hydrolysis. Values

563

are averages of three independent replicates; error bars represent average ± one standard deviation. 28


Page 28 of 39

Page 29 of 39


564

Figure 6 Effect of the C-terminal region deletion on the thermostability at 70 °C (left panel) and

565

expression level, specific activity (right panel) of EGUS. The thermostability and specific activity

566

tests were conducted in triplicate, and the errors represent mean ± one standard deviation. For

567

SDS-PAGE, M: marker; lane 1: EGUS-supernatant; lane 2: EGUS-whole cell; lane 3: D588-603-

568

supernatant; lane 4: D588-603-whole cell; lane 5: D597-603-supernatant; lane 6: D597-603-whole

569

cell. The loading volume of the supernatant and whole cell was 4 μL.

570

Figure 7 Scheme of two-step hydrolysis of glycyrrhizin (GL) into glycyrrhetic acid (GA)

571

mediated by PGUS.

572

Figure 8 Fed-batch process for preparing glycyrrhetinic acid (GA) by glycyrrhizin (GL)

573

hydrolysis mediated by PGUS and mutants D590-604 and D591-604. (a) GA concentration, (b)

574

GL conversion and (c) GA yield as a function of reaction time. For reactions mediated by PGUS,

575

GL was fed at 100 min. For reaction mediated by D590-604 and D591-604, GL was fed at 20 min,

576

140 min and 200 min. Values are averages of three independent replicates; error bars represent

577

average ± one standard deviation.

578

29



579

580

Page 30 of 39

Table 1 Kinetic parameters of PGUS and mutants Enzyme

Km (mM)

kcat (s-1)

kcat/Km (s-1 mM-1)

PGUS D590-604 D591-604 D595-604 D597-604

1.2 ± 0.09 0.5 ± 0.03 1.3 ± 0.1 1.4 ± 0.07 0.6 ± 0.02

162.4 ± 0.6 160.6 ± 8.5 427.1 ± 6.0 313.1 ± 2.2 193.6 ± 1.9

135.3 321.2 328.5 223.6 322.7

Results are from triplicate measurement, and uncertainties are denoted as the average ± one standard deviation.

581 582 583 584 585 586 587 588

30


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Figure 1 192x494mm (600 x 600 DPI)



Figure 2 133x223mm (600 x 600 DPI)


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Figure 3 136x232mm (600 x 600 DPI)



Figure 4 121x103mm (600 x 600 DPI)


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Figure 5 66x31mm (600 x 600 DPI)





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Figure 8 199x499mm (600 x 600 DPI)


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Graphic for table of contents 47x26mm (600 x 600 DPI)


Computation-aided rational deletion of C-terminal region improved the

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