Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria.

Nayuta Furukawa,Akimasa Miyanaga,Masahiro Nakajima,Hayao Taguchi,Misato Togawa

doi:10.1186/s13568-014-0076-1

Nayuta Furukawa, Akimasa Miyanaga + Show 3 more

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https://doi.org/10.1186/s13568-014-0076-1

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Abstract

NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD+. Although non-allosteric d-LDHs from Lactobacilli have been extensively studied, the catalytic properties of allosteric d-LDHs from Gram-negative bacteria except for Escherichia coli remain unknown. We characterized the catalytic properties of d-LDHs from three Gram-negative bacteria, Fusobacterium nucleatum (FNLDH), Pseudomonas aeruginosa (PALDH), and E. coli (ECLDH) to gain an insight into allosteric mechanism of d-LDHs. While PALDH and ECLDH exhibited narrow substrate specificities toward pyruvate like usual d-LDHs, FNLDH exhibited a broad substrate specificity toward hydrophobic 2-ketoacids such as 2-ketobutyrate and 2-ketovalerate, the former of which gave a 2-fold higher kcat/S0.5 value than pyruvate. Whereas the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves below pH 6.5, FNLDH and ECLDH, and PALDH showed marked positive and negative cooperativity, respectively, in the pyruvate saturation curves above pH 7.5. Oxamate inhibited the catalytic reactions of FNLDH competitively with pyruvate, and the PALDH reaction in a mixed manner at pH 7.0, but markedly enhanced the reactions of the two enzymes at low concentration through canceling of the apparent homotropic cooperativity at pH 8.0, although it constantly inhibited the ECLDH reaction. Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg2+ also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH. Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.

Highlights

NAD-dependent D- and L-lactate dehydrogenases (D-LDH, EC 1.1.1.28; and L-LDH, EC 1.1.1.27) reduce pyruvate to D- and L-lactate, respectively, with oxidization of NADH into NAD+, and act at the last step of the glycolytic pathway under anaerobic conditions (Holbrook et al 1975)
D-lactate dehydrogenases (D-LDHs) from Fusobacterium nucleatum (FNLDH), D-LDH from Pseudomonas aeruginosa (PALDH), and D-LDH from Escherichia coli (ECLDH) were stable in the pH ranges of 5.0–8.5, 4.0–8.0, and 5.0–10.0, respectively, during treatment at 30°C for 1 h (Figure 2 a, b, c)
The theoretical molecular weights of FNLDH, PALDH, and ECLDH are 40.0, 37.8, and 38.5 kDa, suggesting that FNLDH and PALDH at that of homotetrameric and homotrimeric structures, respectively, and ECLDH was eluted at the position of homooctameric and homotetrameric structures

Summary

Introduction

NAD-dependent D- and L-lactate dehydrogenases (D-LDH, EC 1.1.1.28; and L-LDH, EC 1.1.1.27) reduce pyruvate to D- and L-lactate, respectively, with oxidization of NADH into NAD+, and act at the last step of the glycolytic pathway under anaerobic conditions (Holbrook et al 1975). DLDHs belong to a large D-2-hydroxyacid dehydrogenase (D-HydDH) superfamily, which comprises various kinds of D-isomer dehydrogenases such as the D-glycerate (Greenler et al 1989; Goldberg et al 1994), D-3-phosphoglycerate (Tobey and Grant 1986; Grant 1989), D-2-hydroxyglutarate (Buckel and Barker 1974; Martins et al 2005), and D-2hydroxyisocaproate (Lerch et al 1989; Dengler et al 1997) dehydrogenases, and even non- D-HydDHs such as the formate (Vinals et al 1993; Lamzin et al 1994; Popov and Lamzin 1994), phosphite (Costas et al 2001; Woodyer et al 2005), and L-alanine (Baker et al 1998; Tripathi and Ramachandran 2008) dehydrogenases These enzymes possess highly divergent primary sequences, their tertiary structures are very similar to one another. Beside the D-LDH from E. coli, the D-3-phosphoglycerate dehydrogenases (D-PgDHs) from some bacteria, such as E. coli (Tobey and Grant 1986; Grant 1989; Schuller et al 1995) and Mycobacterium tuberculosis (Dey et al 2005), are only known as allosteric and homotetrameric enzymes in the D-HydDH superfamily

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