Essential role of amino acid position 71 in substrate preference by meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum IAM14863

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Highlights

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    The first report on the roles of non-active site of StDAPDH and meso-DAPDH family.

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    Mutation destroyed the cation-π interaction between R71 and Y205 of StDAPDH.

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    CgA69R was created with CgDAPDH as template for reverse verification.

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    CgA69R showed higher catalytic efficiencies of amination toward 2-keto acids.

Abstract

meso­-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible oxidative deamination of the d­configuration of meso-2,6­diaminopimelate (meso-DAP) and is thought to have substrate specificity toward meso-DAP. The discovery of the meso-DAPDH from Symbiobacterium thermophilum IAM14863 (StDAPDH) revealed meso-DAPDH members with broad substrate specificity. In order to elucidate the substrate-preference mechanism of StDAPDH, it is necessary to identify the key residues related to this mechanism. Our previous work suggested that the non-active-site R71 of StDAPDH was related to substrate preference. Here, we report the key roles of the non-active site on the catalysis of StDAPDH. In order to explore the mechanism through which non-active-site R71 only affected the amination activity of StDAPDH, we performed molecular dynamic simulations and investigated the functional role of R71 in the type II meso-DAPDH StDAPDH. Site-directed mutagenesis with the allelic site A69 of CgDAPDH as a target proved that when replaced by Arg at position 71 of StDAPDH, the CgA69R mutant showed higher catalytic efficiencies toward a series of 2-keto acids, ranging from 1.2- to 1.5-fold. These findings provide some guidelines for improving our understanding of the broad substrate specificity of StDAPDH.

Introduction

meso­-Diaminopimelate dehydrogenase (EC 1.4.1.16, meso-DAPDH) is a NADP+-dependent enzyme that catalyzes the reversible oxidative deamination of the d-configuration of meso-2,6-­diaminopimelate (meso-DAP) to produce l­2­amino­-6-oxopimelate [[1], [2], [3], [4], [5]]. Traditionally, meso-DAPDH was thought to only catalyze the oxidative deamination of meso-DAP with high substrate specificity and stereoselectivity. However, with the identification of the meso-DAPDH from Symbiobacterium thermophilum IAM14863 (StDAPDH) [6], it was recognized that there are also naturally occurring meso-DAPDH which can catalyze the reverse reductive amination reaction to yield d-amino acids. Therefore, we wondered whether there were other meso-DAPDH proteins in nature that could catalyze reductive amination reactions, similar to StDAPDH. In our previous work [7], the current DAPDHs from the RefSeq database were divided into two types [7]; type I, represented by the meso-DAPDH from Corynebacterium glutamicum ATCC13032 (CgDAPDH) [[8], [9]], can only catalyze the oxidative deamination of meso-DAP with high catalytic activities, whereas type II, represented by StDAPDH, shows broader substrate specificity than type I DAPDHs toward 2-keto acids.

Crystal structure comparisons and evolutionary conservation analyses have indicated that catalytic residues from type I and type II DAPDHs are the same and are highly conserved, except for Phe146/Trp144 (StDAPDH/CgDAPDH) and Met152/Gln150 [6]. Site-directed mutagenesis has shown that Phe146 of StDAPDH does not affect substrate binding, but does decrease catalytic efficiency [6]. Additionally, Met152 of StDAPDH has been shown to be a key residue in substrate binding, owing in part to its location around two entrance tunnels of pyruvic acid [10]. Both Phe146 and Met152 are not key residues discriminating between the reductive amination abilities of StDAPDH and CgDAPDH. In our previous work [7], Arg71 (R71) of StDAPDH was found to be a substrate preference-related residue by site-directed mutagenesis. Moreover, considering its high conservation, this site may be an indicator of the amination preference of type II molecules. R71 is also a non-active site residue and is located next to the NADP(H)-binding site but not in the substrate binding site [7]. Moreover, at present, studies on the catalytic mechanisms of meso-DAPDHs have been limited to the analysis of several members, including CgDAPDH, StDAPDH [10], the meso-DAPDH from Clostridium tetani E88 (CtDAPDH) [11], and the meso-DAPDH from Ureibacillus thermosphaericus (UtDAPDH) [12]. All of these studies have revealed the modes of substrate binding and entrance for these meso-DAPDHs; however, the roles of non-active-site residues of meso-DAPDHs have not yet been determined.

Therefore, in this study, we aimed to expand on our previous work to illustrate the mechanisms through which the non-active-site R71 was responsible for the substrate specificity of StDAPDH using molecular dynamic simulations and further verified whether the results were indicators of the amination of meso-DAPDH by reverse mutagenesis.

Section snippets

Materials

DNA polymerase KOD plus-neo was ordered from TOYOBO (Osaka, Japan). NADPH and NADP+ were ordered from Codexis (Redwood City, CA, USA). Pyruvic acid and meso-DAP were purchased from Tokyo Chemical Industry (Tokyo, Japan).

Site-directed mutagenesis

Whole-plasmid polymerase chain reaction was performed to create all variants following the protocols reported previously [6]. Overexpression and purification were performed as previously described [7].

Kinetic constants assay

Analysis of kinetic constants was performed as previously described [7],

Effects of amino acid substitution at position 71 of StDAPDH on catalytic efficiency

In our previous work [7], when R71 of StDAPDH was replaced with Ala, the catalytic efficiency of amination, not deamination, was dramatically affected (Table 1). In order to investigate the requirements for the type of amino acids that could occupy this position, site-directed mutagenesis was employed to create five single mutants with different types of sidechains at position 71. The kinetic parameters of mutant and wild-type proteins toward pyruvic acid and meso-DAP are listed in Table 1.

Conclusions

In this study, the roles of the non-active site R71 on the catalysis of meso-DAPDH from Symbiobacterium thermophilum IAM14863 (StDAPDH) were examined using molecular dynamic simulations. Compared with the deamination reaction of StDAPDH, for amination, pyruvic acid binding resulted in different secondary structures for the region encompassing R71. Moreover, there was a cation-π interaction between R71 and Y205 when pyruvic acid was used as a substrate; mutation of R71 of SDAPDH destroyed the

Author contributions

Ya’nan Zhang planned the project, analyzed kinetic parameters, and drafted the manuscript. Qinyuan Ma performed the molecular dynamics simulation and helped to draft the manuscript. Miaomiao Dong constructed all variants and performed protein purification. Xianhai Zhang and Yichu Chen analyzed the kinetic parameters. Xiuzhen Gao contributed to evaluation of the results, supervised the project, and proofread the manuscript. Yanda Song revised the manuscript. All authors read and approved the

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgement

This work was financially supported by the National Natural Science Foundation of China (grant no. 21402109) and China Postdoctoral Science Foundation (grant no. 2016M592191).

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