Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria
AMB Express volume 4, Article number: 76 (2014)
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.
NAD-dependent d- and l-lactate dehydrogenases (d-LDH, EC 220.127.116.11; and l-LDH, EC 18.104.22.168) 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. ). Although both types of enzymes catalyze identical reactions except for the distinct chirality of the lactate product, they are evolutionarily distinct from each other (Taguchi and Ohta ; Bernard et al. ; Kochhar et al. ). d-LDHs 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. ; Goldberg et al. ), d-3-phosphoglycerate (Tobey and Grant ; Grant ), d-2-hydroxyglutarate (Buckel and Barker ; Martins et al. ), and d-2-hydroxyisocaproate (Lerch et al. ; Dengler et al. ) dehydrogenases, and even non- d-HydDHs such as the formate (Vinals et al. ; Lamzin et al. ; Popov and Lamzin ), phosphite (Costas et al. ; Woodyer et al. ), and l-alanine (Baker et al. ; Tripathi and Ramachandran ) dehydrogenases. Although these enzymes possess highly divergent primary sequences, their tertiary structures are very similar to one another.
In the case of l-LDHs, it is known that allosteric types of the enzyme are widely distributed in bacteria including Lactobacilli (Garvie ). These enzymes commonly require fructose 1,6-bisphosphate (FBP) to exhibit high catalytic activity, and usually exhibit sigmoidal shaped substrate saturation curves unless FBP is present. l-LDHs generally have tetrameric structures comprising four identical subunits, and allosteric l-LDHs undergo cooperative structural changes in the substrate and FBP binding sites through quaternary structural changes (Iwata et al. ; Arai et al. ). On the other hand, no allosteric property has been reported for Lactobacillus d-LDHs. The Lactobacillus d-LDHs consistently have a dimeric structure comprising identical subunits, and exhibit hyperbolic shaped saturation curves for substrate pyruvate. Interestingly, it was reported that d-LDH from E. coli exhibits a sigmoidal pyruvate saturation curve, which is changed to the hyperbolic one by 2-ketobutyrate (Tarmy and Kaplan ). The reported results of size-exclusion chromatography suggested that this enzyme has a homotetrameric structure, although the detailed catalytic properties and 3D structure of the enzyme remain unknown. Beside the d-LDH from E. coli, the d-3-phosphoglycerate dehydrogenases (d-PgDHs) from some bacteria, such as E. coli (Tobey and Grant ; Grant ; Schuller et al. ) and Mycobacterium tuberculosis (Dey et al. ), are only known as allosteric and homotetrameric enzymes in the d-HydDH superfamily.
In this paper, we describe the detailed biochemical analysis of allosteric d-LDHs from three Gram-negative bacteria; Fusobacterium nucleatum subsp. nucleatum, an obligate anaerobe; Pseudomonas aeruginosa, an obligate aerobe; and E. coli, a facultative anaerobe, demonstrating that bacterial d-LDHs have highly divergent catalytic properties.
Materials and methods
Cloning, expression, and purification
Genomic DNA of F. nucleatum subsp. nucleatum JCM14847 (=ATCC25586) and P. aeruginosa JCM8532 (=PAO1) was purchased from the Riken BioResource Center (Japan). Genomic DNA of E. coli BL21(DE3) was isolated by ISOGEN (NIPPON GENE, Tokyo, Japan). The genes encoding the FN0511 (FNLDH), PA0927 (PALDH), and ECD_01352 (ECLDH) proteins (Genbank accession numbers: AAL94707.1, AAG04316.1, and ACT43236.1, respectively) were amplified via PCR using genomic DNA as templates. The primer pairs used were, 5'-CGCTCGAGATGCAAAAAACTAAGATAATATTTTTTG-3' and 5'-GGGATCCATTTATTGATTTTGTGGAACTTC-3' for the fn0511 gene, 5'-CGCATATGCGCATCCTGTTCTTCAGCAG-3' and 5'- CGGGATCCTCAGGCCCGGACCCGATTG-3' for the pa0927 gene, and 5'-CGCATATGAAACTCGCCGTTTATAGC-3' and 5'-CGGGATCCTTAAACCAGTTCGTTCGGGC-3' for the ecd_01352 gene. The amplified fn0511, pa0927, and ecd_01352 genes were inserted into T-vector pMD20 (TaKaRa Bio, Shiga, Japan). After purification of the plasmids carrying these genes, the plasmids were digested with restriction enzymes and the genes were inserted into pCold I (TaKaRa Bio). E. coli Rosetta2 (DE3) (Merck Millipore, Darmstadt, Germany) was transformed using the constructed plasmids and cultured at 37°C in 2 × YT medium (1.6% tryptone, 1.0% yeast extract and 0.5% NaCl) containing 100 μg/ml ampicillin. After the optical density of the culture at 600 nm (OD600) had reached 0.6, protein expression was induced using 0.5 mM isopropyl-β-1-d-thiogalactopyranoside at 15°C overnight. The harvested cells were lysed by sonication in 50 mM HEPES-NaOH buffer (pH 8.0) containing 150 mM NaCl and 10 mM imidazole on ice. After each sample had been centrifuged at 27,000 × g, the supernatant was applied to HisTrap FF crude column (5 ml) (GE Healthcare, Buckinghamshire, UK), and was then eluted with a linear gradient of 10-250 mM imidazole. After dialysis against 50 mM HEPES-NaOH buffer (pH 8.0), the protein solution was further purified using a UnoQ column (Bio-Rad Laboratories, California, USA). The target protein was eluted with a linear gradient of 0-300 mM NaCl. The buffer for FNLDH and ECLDH was changed to 5 mM HEPES-NaOH buffer (pH 8.0), and the buffer for PALDH to 5 mM sodium acetate buffer (pH 5.0) using Amicon Ultra 30,000 molecular weight cut-off (Merck Millipore). The concentrations of FNLDH, PALDH, and ECLDH were determined spectrophotometrically at 280 nm using theoretical extinction coefficients of 24,215, 19,285, and 17,140 M-1 cm-1, respectively (Pace et al. ).
The pufiried enzymes were separated on a 11% (w/v) SDS-polyacrylamide gel (Laemmli ). Approximately 5 μg of the enzymes were loaded as 5% (v/v) glycerol solutions containing 1% (w/v) SDS, 2.5% (v/v) β-mercaptoethanol and 0.05% bromophenol blue. Precision Plus Protein Unstained Standards (Bio-Rad Laboratories) was used as molecular weight markers. Proteins were stained with Coomassie Brilliant Blue.
Enzyme activity was determined by measuring the reduction of the absorbance at 340 nm derived from NADH for 1 minute. The reactions were performed at 30°C in various 50 mM buffers containing 0.1 mM NADH and a substrate. The data that showed significant cooperative effects of the substrate were interpreted using the Hill equation (Equation 1) (Dixon and Webb ).
where v is the reaction velocity, [S] the ligand concentration, such as pyruvate, FBP, or Mg2+, kcat the turnover rate of catalysis, vmin the reaction velocity with no ligand, S0.5 the half-saturation concentration of a ligand, and nH the Hill coefficient. Kinetic parameters were obtained by curve fitting of the data with KaleidaGraph ver 3.51. The data that indicated significant substrate inhibition were interpreted using the equation for substrate inhibition (Equation 2) (Eszes et al. ).
where Ki is the inhibition constant. The kinetic parameters for oxamate at pH 7.0 were calculated by curve fitting of the data with GraFit version 7.0.3. The data that indicated significant competitive-type and mix-type inhibition were interpreted using the equation for competitive-type (Equation 3) and mix-type inhibition (Equation 4), respectively.
where Km is the Michaelis constant, and Ki' the inhibition constant. The data of oxamate inhibition for FNLDH, PALDH, and ECLDH at pH 8.0 were interpreted using the equation for competitive-type (Equation 5), mix-type (Equation 6), and non-competitive-type (Equation 7) inhibition of allosteric enzyme, respectively.
where kcat' is the turnover rate of catalysis in the presence of oxamate, Kact the half-saturation concentration of oxamate for activation, and nH' the hill coefficient for oxamate.
Temperature and pH stability
The pH-stability was determined by measuring the remaining activity after incubation of the enzymes (1 μM) in various 50 mM buffers at 30°C for 1 h. The thermostability was determined by measuring the remaining activity after incubation of FNLDH (1 μM) or ECLDH (1 μM) in 50 mM HEPES-NaOH buffer (pH 8.0), or of PALDH (1 μM) in 50 mM sodium acetate buffer (pH 5.0) at various temperatures for 30 min.
Each protein solution diluted to 1 mg/ml (1 ml) was applied to Superdex 200 (Hiload 16/60; GE Healthcare) equilibrated with 50 mM buffer containing 150 mM NaCl. HEPES-NaOH (pH 8.0) was used for FNLDH and ECLDH, and sodium acetate buffer (pH 5.0) for PALDH. Ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) (GE Healthcare) were used as standard proteins. Blue dextran 2000 (2,000 kDa; GE Healthcare) was used to determine the void volume of the column.
The recombinant FNLDH, PALDH and ECLDH consistently exhibited marked catalytic activity toward pyruvate, and were successively purified to homogeneous protein samples (Figure 1a). FNLDH, PALDH, and 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). FNLDH, PALDH, and ECLDH were stable up to 39, 57, and 49°C, respectively, under favorable pH conditions, i.e., pH 8.0 for FNLDH and ECLDH, and pH 5.0 for PALDH (Figure 2d).
The purified FNLDH and PALDH samples each gave a single protein peak on size-exclusion chromatography analysis, and exhibited apparent molecular weights of 160 and 120 kDa, respectively (Figure 1b). On the other hand, the ECLDH sample gave two peaks corresponding to molecular weights of 330 and 150 kDa. 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. Since it has been reported that native d-LDH from E. coli cells gives only a peak that corresponds to the molecular weight of 130 kDa (Tarmy and Kaplan ), the apparently octameric form of ECLDH is likely an artificial product due to the overexpression, and exhibited slightly but significantly lower specific activity than the tetrameric form (data not shown). Therefore, only homotetrameric ECLDH was used for the detailed analysis below.
pH-Dependency of kinetic parameters
The kinetic parameters, kcat, S0.5, kcat/S0.5, and nH values, were determined by pyruvate reduction assaying in the pH range of 4.5-9.0 (Figure 3). The three enzymes consistently showed virtually constant kcat values independently of pH, and PALDH and ECLDH exhibited about 5-fold larger kcat values than FNLDH. The three enzymes also consistently showed constant pyruvate S0.5 values below pH 7.0, and PALDH and FNLDH exhibited approximately one order of magnitude smaller S0.5 values than ECLDH. Above pH 8.0, the three enzymes exhibited increased S0.5 values, depending on the pH, and their kcat/S0.5 values changed mostly according to the changes in the S0.5 values. ECLDH apparently showed lower pH-dependence of the S0.5 value than the other two enzymes, of which PALDH showed slightly higher pH-dependence than FNLDH. Under acidic conditions below pH 6.0, the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves, and no marked cooperativity in pyruvate binding. Under higher pH conditions, however, FNLDH and ECLDH showed markedly sigmoidal saturation curves for pyruvate, i.e., positive homotropic cooperativity, giving maximal nH values of about 2.0. In contrast, PALDH showed negative homotropic cooperativitiy above pH 8.0, giving a minimal nH value of 0.5.
The kinetic parameters of the d-LDHs for various 2-ketoacids are summarized in Table 1. For pyruvate, PALDH exhibited the highest catalytic efficiency among the three enzymes, since it exhibited a small S0.5 like FNLDH and a large kcat like ECLDH. ECLDH showed a narrow substrate specificity toward pyruvate among hydrophobic 2-ketoacid substrates, although ECLDH consistently exhibited relatively high catalytic activity toward oxaloacetate, glyoxylate and hydroxypyruvate. PALDH showed a similar substrate specificity to that of ECLDH, exhibiting relatively high catalytic activity toward oxaloacetate, glyoxylate and hydroxypyruvate, although it generally exhibited higher catalytic efficiencies for these substrates than ECLDH. In contrast, FNLDH showed a broad substrate specificity toward relatively bulky hydrophobic 2-ketoacids, and exhibited particularly high catalytic activity toward 2-ketobutyrate and 2-ketovalerate, for which the catalytic efficiencies were comparable to that for pyruvate. FNLDH also showed relatively high activity to oxaloacetate and hydroxypyruvate, but poor activity toward glyoxylate.
Inhibition and activation by oxamate
Oxamate, an inert pyruvate analogue, inhibited the reactions of the three enzymes at pH 7.0, where the enzymes showed no marked homotropic cooperativity, in different manners, i.e. an apparently competitive manner with pyruvate for FNLDH, and a mixed manner for PALDH and ECLDH (Figure 4). This suggests that oxamate is bound not only to the catalytic site, but also to unknown allosteric sites in PALDH and ECLDH, whereas it is bound mostly to the catalytic site in FNLDH. Oxamate apparently enhanced the reactions of FNLDH and PALDH, and exhibited the highest activating effects at 20 mM and 2.5 mM, respectively (Figure 5a, b), whereas oxamate inhibited the ECLDH reaction (Figure 5c), at pH 8.0, where the three enzymes exhibited marked homotropic cooperativity. FNLDH and PALDH showed no significant homotropic (positive or negative) cooperativity in the presence of 20 mM and 2.5 mM oxamate, respectively (Figure 5d, e). In ECLDH, the inhibition occurred in a noncompetitive manner toward pyruvate (Figure 5f), suggesting that it is mostly bound to the allosteric site of ECLDH at pH 8.0.
Heterotropic activation of allosteric d-LDHs
Bacterial allosteric l-LDHs are commonly activated by FBP, which usually induces drastic improvement of the substrate S0.5 values of the enzymes (Garvie ). In addition, Lactobacillus casei l-LDH requires some divalent metal ions (e.g., Mn2+) (Arai et al. ), and the Thermus caldophilus enzyme is activated also by citrate under slightly acidic conditions (Taguchi et al. ). We therefore evaluated the effects of FBP, citrate and divalent metal ions at pH 8.0 in the presence of the S0.5 pyruvate. FBP and citrate (1.0 mM) slightly enhanced the catalytic reactions of the three d-LDHs, and divalent metal ions such as Mg2+ and Mn2+ more markedly enhanced the reactions of FNLDH and PALDH (Figure 6a). In the case of FNLDH, Mg2+ and FBP showed significant activation effects, and Mg2+ gave a 2.8-fold smaller S0.5 value (1.5 mM) than FBP (4.2 mM) for the enzyme activation (Figure 6 and Table 2). In the presence of 10 mM FBP or 5 mM Mg2+, FNLDH exhibited hyperbolic pyruvate saturation curves, the pyruvate S0.5 (Km) value being reduced by approximately 1.4-fold or twice, respectively (Figure 6, and Table 3). For PALDH, Mg2+ and FBP also showed activation effects, and Mg2+ gave a 75-fold smaller S0.5 value (0.16 mM) than FBP (12 mM) for the enzyme activation (Figure 6 and Table 2). In this case, FBP and Mg2+ also consistently reduced the pyruvate S0.5 value significantly, and the negative cooperativity in pyruvate binding (Figure 6, and Table 3). In the case of ECLDH, FBP and Mg2+ exhibited only slight activation effects on the enzyme reaction, and the latter even markedly inhibited the reaction at high concentrations (Figure 6, and Tables 2 and 3).
Kinetic profiles for NADH
The apparent kinetic parameters for NADH were determined at the saturation concentration of pyruvate at pH 7.0 and 8.0 (Table 3). FNLDH and PALDH exhibited virtually the same apparent NADH S0.5 value (about 0.035 mM), which was approximately twice smaller than that of ECLDH. Since the pyruvate S0.5 values were determined at 0.1 mM NADH (Figure 3 and Table 3), these values might be larger than the real values because of an insufficient concentration of NADH, particularly in the case of ECLDH. PALDH and ECLDH exhibited hyperbolic NADH saturation curves independently of pH, FBP or Mg2+. On the other hand, FNLDH exhibited a sigmoidal one at pH 8.0, and exhibited an nH value of 1.6, which was slightly reduced by FBP or Mg2+.
The characteristics of FNLDH, PALDH and ECLDH demonstrate the high variety of the functions of bacterial d-LDHs. Of the three enzymes, PALDH exhibits a markedly smaller pyruvate S0.5 value than ECLDH, and a larger kcat value than FNLDH at pH 7.0 (Table 3). Although the crucial role of PALDH in the metabolism of P. aeruginosa remains uncertain, the enzyme is likely involved in regulation of the NAD+/NADH ratio within the cells like the heart-type l-LDH isozymes of aerobic tissues (Holbrook et al. ). On the other hand, ECLDH with the large S0.5 is likely favorable for facultative anaerobes such as E. coli, in which the pyruvate level within the cells dynamically changes between anaerobic and aerobic conditions like the muscle-type l-LDH isozymes (Holbrook et al. ). FNLDH exhibits a small pyruvate S0.5 value although F. nucleatum is obligate anaerobe. In F. nucleatum cells, pyruvate is likely consumed mostly through pathways other than lactate fermentation, such as butyrate fermentation (Kapatral et al. ), and therefore pyruvate within the cells may be maintained at low concentrations. It is particularly notable that FNLDH prefers relatively bulky hydrophobic 2-ketoacids, such as 2-ketobutyrate and 2-ketovalerate, which give apparently equivalent or even higher kcat/Km value than pyruvate, unlike in the cases of the other two enzymes (Table 1). This substrate preference suggests that FNLDH might be used for not only pyruvate reduction but also the reduction or oxidation of other bulky 2-ketoacid or 2-hydroxy acids, respectively, although the actual physiological substrates of the enzyme remains uncertain.
FNLDH apparently has a size-preference for 2-ketoacid substrates intermediate between those of conventional d-LDH and d-2-hydroxyisocaproate dehydrogenase (Hummel et al. ). Like usual d-LDHs, nevertheless, FNLDH has aromatic residues, Phe52 and Tyr299 (residue numbers are according to d-LDH from Lactobacillus pentosus), which are proposed to play key roles in the substrate recognition in the binding pocket for the substrate C3 side chains (Tokuda et al. ; Ishikura et al. ). It is also noteworthy that FNLDH exhibits relatively high activity toward 2-ketoisovalerate since little is known as to an enzyme that exhibits high catalytic activity toward C3-branched 2-ketoacid substrates in the d-LDH-related d-HydDH family. It is known that d-mandelate dehydrogenase from Enterococcus faecalis exhibits high catalytic activity toward hydrophobic C3-branched ketoacids such as 2-ketoisovalerate and benzoyl formate (Tamura et al. ). Nevertheless, this enzyme belongs to another d-HydDH family, the 2-ketopantate reductase-related family, which is evolutionally separate from the d-LDH-related family (Wada et al. ; Miyanaga et al. ). Hence, FNLDH is the first case of a d-LDH-related d-HydDH that exhibits relatively high catalytic activity toward C3-branched 2-ketoacid substrates and may be useful for enzymatic production of C3-branched d-2-hydroxyacids.
For FNLDH, oxamate inhibits the reaction competitively with pyruvate at pH 7.0, and substitutes for the role of pyruvate in the homotropic enzyme activation at pH 8.0, converting the sigmoidal saturation curve for pyruvate to a hyperbolic one (Figure 5d). These effects indicate that oxamate simply competes with pyruvate for a common binding site, i.e., the catalytic site in FNLDH. It is likely that PALDH binds oxamate at both the catalytic and allosteric sites, since oxamate inhibits the reaction in a mixed manner at pH 7.0 (Figure 4), as in the case of ECLDH, but stimulates the reaction at low concentration at pH 8.0, where oxamate likely exhibits an activation effect mostly through binding to the catalytic site and inhibitory effects mostly through binding to the unknown allosteric site (Figure 5b,e). In the case of ECLDH, it was suggested previously that oxamate is likely bound mostly to a binding site other than the catalytic site (Tarmy and Kaplan ), this being consistent with the results of this study (Figure 4c). These results indicate that the three enzymes differ in their allosteric behaviors, and specificities to allosteric effectors, although the actual allosteric binding sites or physiological effectors of these enzymes remain uncertain.
FNLDH and PALDH are the first cases of d-LDHs that are activated by FBP, which is a common activator for bacterial allosteric l-LDHs (Garvie ). It is nevertheless unlikely that FBP is really the specific activator of the two d-LDHs, since the two enzymes require apparently higher concentrations of FBP than the physiological one for their activation (Figure 6b,c and Table 2). On the other hand, the Mg2+ ion is more effective than FBP as to activation of the two d-LDHs, particularly PALDH (Figure 6, and Tables 2 and 3). It is, however, also unlikely that the Mg2+ ion is the specific activator of these enzymes, since some other divalent metal ions such as Sr2+ and Mn2+ also improve their reactions (Figure 6a). These two enzymes thus appear to be activated through the comprehensive effects of several activators with low specificities, physiologically, although it is possible that they have unknown activators with higher specificities. In the case of ECLDH, FBP and Mg2+ only slightly enhance the catalytic reaction, and the latter even markedly reduces the reaction at higher concentrations (Figure 6d,g and Table 3). We have evaluated other possible effectors, such as l-alanine, l-glutamate and l-aspartate, for this enzyme, but have not found out any potential activator for ECLDH (data not shown). This implies that ECLDH is only negatively controlled under alkaline conditions.
The allosteric and tetrameric d-PgDHs from E. coli and M. tuberculosis were extensively studied as to their structure-function relationship (Tobey and Grant ; Grant ; Schuller et al. ; Dey et al. ; Grant ). The d-PgDHs have V-type regulatory mechanisms, in which l-serine inhibits the enzyme reaction in a non-competitive manner, whereas the three d-LDHs apparently exhibit K-type rather than V-type regulation. These d-PgDHs have additional functional domain(s), ACT domain and ASB domain, which undergo not only interactions with allosteric effector, l-serine, but also inter-subunit interactions with each other for the construction of tetrameric structure. In contrast, d-LDHs have no additional protein domain. The three d-LDHs from Gram-negative bacteria thus greatly differ from these d-PgDHs in both regulatory mechanism and protein structure.
It is reported that bacterial allosteric l-LDHs consistently undergo the Monod-Wyman-Changeux-type (MWC-type: pre-existing type) (Monod et al. ; Iwata et al. ; Arai et al. ) allosteric transition, where the active (R) state with high affinity to pyruvate and FBP, and the inactive (T) state with low affinity. However, such drastic structural changes appear to be unnecessary for the three d-LDHs, which exhibit apparently much smaller changes in catalytic activity due to the allosteric transition than the l-LDHs. In fact, the MWC model is not available for the case of PALDH, which shows negative homotropic cooperativity. In addition, FNLDH shows significant positive homotropic cooperativity also for NADH binding, which is less affected by FBP or Mg2+ than pyruvate binding (Table 3), suggesting that FNLDH changes in its structure through two steps, the NADH binding and following pyruvate binding steps, in the allosteric transition. The Lactobacillus d-LDHs consistently have dimeric structure, whereas the three d-LDHs from Gram-negative bacteria have tetrameric structure. Thus, tetramerization might be correlated with the allostery of d-LDHs. It is highly desirable to determine the 3D-structures of these d-LDHs.
NAD-dependent d-lactate dehydrogenase
d-LDH from Escherichia coli
d-LDH from Fusobacterium nucleatum
d-LDH from Pseudomonas aeruginosa
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This work was partially supported by a Grant-in-Aid (No. 23580120 to H.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).
The authors declare that they have no competing interests.
NF designed the study, carried out most of the biochemical studies and drafted the manuscript. AM designed the study and helped to draft the manuscript. MT participated in the kinetics analysis of PALDH. MN participated in the design of the study and the kinetics analysis, and helped to draft the manuscript. HT conceived and designed the study, and helped to draft the manuscript. All authors read and approved the final manuscript.
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Furukawa, N., Miyanaga, A., Togawa, M. et al. Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria. AMB Expr 4, 76 (2014). https://doi.org/10.1186/s13568-014-0076-1