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

Furukawa N, Miyanaga A, Togawa M, Nakajima M, Taguchi H - AMB Express (2014)

Bottom Line: NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD(+).Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg(2+) 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Chiba, Japan.

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 k cat/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 Mg(2+) 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.

No MeSH data available.


Related in: MedlinePlus

pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH (N,N-Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH (N-cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH (N-cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.
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Figure 2: pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH (N,N-Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH (N-cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH (N-cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.

Mentions: 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).


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

Furukawa N, Miyanaga A, Togawa M, Nakajima M, Taguchi H - AMB Express (2014)

pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH (N,N-Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH (N-cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH (N-cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230899&req=5

Figure 2: pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH (N,N-Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH (N-cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH (N-cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.
Mentions: 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).

Bottom Line: NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD(+).Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg(2+) 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Chiba, Japan.

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 k cat/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 Mg(2+) 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.

No MeSH data available.


Related in: MedlinePlus