Determinants of dual substrate specificity revealed by the crystal structure of homoisocitrate dehydrogenase from Thermus thermophilus in complex with homoisocitrate·Mg2+·NADH

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Highlights

  • Crystal structure of HICDH in a complex with HIC/Mg2+/NADH was determined.

  • Mechanism of the dual substrate specificity was elucidated.

  • The structure provided an insight into the evolution of substrate specificity of HICDH family.

Abstract

HICDH (Homoisocitrate dehydrogenase) is a member of the β-decarboxylating dehydrogenase family that catalyzes the conversion of homoisocitrate to α-ketoadipate using NAD+ as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the α-aminoadipate pathway. Although typical HICDHs from fungi and yeast exhibit strict substrate specificities toward homoisocitrate (HIC), HICDH from a thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both HIC and isocitrate (IC) as substrates at similar efficiencies. We herein determined the crystal structure of the quaternary complex of TtHICDH with HIC, NADH, and Mg2+ ion at a resolution of 2.5 Å. The structure revealed that the distal carboxyl group of HIC was recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. Model structures were constructed for TtHICDH in complex with IC and also for HICDH from Saccharomyces cerevisiae (ScHICDH) in complex with HIC. TtHICDH recognized the distal carboxyl group of IC by Arg85 in the model. In ScHICDH, the distal carboxyl group of HIC was recognized by the side chains of Ser98 and Ser108 from one subunit and Asn208 from another subunit of a dimer unit. By contrast, in ScHICDH, which lacks an Arg residue at the position corresponding to Arg85 in TtHICDH, these residues may not interact with the distal carboxyl group of shorter IC. These results provide a molecular basis for the differences in substrate specificities between TtHICDH and ScHICDH.

Introduction

Homoisocitrate dehydrogenase (HICDH) is an enzyme involved in the lysine biosynthetic pathway (α-aminoadipate pathway) of yeast, fungi, and some kinds of bacteria and archaea [1], [2], [3]. HICDH catalyzes the decarboxylating dehydrogenation of homoisocitrate (HIC) into α-ketoadipate (Fig. 1). HICDH is a member of the β-decarboxylating dehydrogenase family, which consists of 3-isopropylmalate dehydrogenase [IPMDH; EC 1.1.1.85] for leucine biosynthesis [4], isocitrate dehydrogenase [ICDH; EC 1.1.1.42] for the tricarboxylic acid (TCA) cycle [5], and HICDH [EC 1.1.1.87] for lysine biosynthesis via α-aminoadipate [6]. Since homologous enzymes are considered to have evolved from a common ancestral enzyme into specialized enzymes [7], [8], [9], [10], [11], comparisons of recognition mechanisms between each enzyme provide useful information on how these homologous enzymes acquired the separate functions in the course of evolution.

Lysine is known to be synthesized via the diaminopimelate (DAP) pathway in bacteria, and via the α-aminoadipate (AAA) pathway in fungi and yeast. We previously found that Thermus thermophilus, which is an extremely thermophilic bacterium, synthesized lysine via AAA [12], cloned the biosynthetic gene cluster, and characterized most of the enzymes involved in the pathway [13], [14], [15], [16], [17], [18], [19]. Some of these enzymes catalyze not only the reaction in lysine biosynthesis but also those of related pathways such as branched-chain amino acid biosynthesis, the TCA cycle, and/or arginine biosynthesis [13], [14], [15], [17]. HICDH from T. thermophilus (TtHICDH) is an enzyme that recognizes HIC and IC as substrates [16] (Fig. 1). Since ancestral enzymes are considered to have been promiscuous and capable of catalyzing reactions for multiple related reactions, TtHICDH may retain the characteristic features of its ancestral enzyme. By contrast, HICDHs from yeast, Saccharomyces cerevisiae (ScHICDH) and Schizosaccharomyces pombe (SpHICDH), distinguish HIC from IC and exhibit strict specificity toward HIC [20], [21]. The substrate specificities of β-decarboxylating dehydrogenase family members appeared to be determined by a few amino acid residues in the loop β3-α4 and the N-terminal region of the following α4 helix [21]. Substrate specificity was converted by amino acid replacement in the corresponding regions of IPMDH and ICDH. We also showed that Arg85 on the α4 helix of TtHICDH is a crucial determinant for substrate specificity and plays a key role in the dual functions of TtHICDH as HICDH and ICDH in a site-directed mutagenesis study [16]. Although determinants of substrate specificity have been proposed for TtHICDH and SpHICDH based on crystallographic analyses coupled with site-directed mutagenesis, the molecular mechanisms underlying substrate recognition by HICDHs has not yet been elucidated due to the lack of substrate-bound structures in most cases [22], [23], [24]. In the present study, we determined the crystal structure of a quaternary complex of TtHICDH binding HIC, NADH, and Mg2+, which revealed the structural feature of TtHICDH exhibiting dual substrate specificity, and also provided structural basis that defined the strict substrate preference of ScHICDH for HIC using its modeled structure.

Section snippets

Protein preparation

We constructed an expression system for TtHICDH as follows. The plasmid pET-tHICDH101 for the expression of TtHICDH which was constructed in our previous study [16] was introduced into Escherichia coli BL21(DE3)CodonPlus-RIL cells. E. coli cells harboring pET-tHICDH101 were grown in 2 × YT medium (1.6% tryptone, 1% yeast extract, and 0.5% NaCl) supplemented with kanamycin (50 μg ml−1) and chloramphenicol (30 μg ml−1) at 37 °C to an optical density of approximately 0.6 at 600 nm. Gene expression

Results and discussion

Crystal structure of TtHICDH in complex with HIC, NADH, and Mg2+ – The crystal structure of TtHICDH complexed with HIC, Mg2+, and NADH (TtHICDH·HIC·Mg2+·NADH) was determined by molecular replacement using the apo-form of TtHICDH (PDB ID 1X0L) at a resolution of 2.5 Å. There were four monomers in the crystallographic asymmetric unit (Fig. 2A). Models were built from Ala2 to Leu334 for each chain. The structure of the four chains were very similar to each other with a root mean square deviation

Author contributions

K. T. performed expression, purification, and biochemical analyses as well as crystallization experiments. K. T. and T. T. performed the crystallographic analysis. K. T., T. T., and M.N. analyzed the 3D structure. K. T., T. T., T. K., and M.N. planned the experiments, and wrote the manuscript.

Acknowledgments

This work was supported in part by JSPS KAKENHI (Grant No. 24228001 to M.N). We are grateful to the staff of the Photon Factory for their assistance with data collection, which was approved by the Photon Factory Program Advisory Committee (Proposal no. 2011G524, 2013G618).

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