Author:
Furukawa Nayuta,Miyanaga Akimasa,Togawa Misato,Nakajima Masahiro,Taguchi Hayao
Abstract
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 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.
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Biophysics
Reference40 articles.
1. Arai K, Ishimitsu T, Fushinobu S, Uchikoba H, Matsuzawa H, Taguchi H: Active and inactive state structures of unliganded Lactobacillus casei allosteric L -lactate dehydrogenase. Proteins 2010, 78: 681–694. doi: 10.1002/prot.22597
2. Arai K, Ichikawa J, Nonaka S, Miyanaga A, Uchikoba H, Fushinobu S, Taguchi H: A molecular design that stabilizes active state in bacterial allosteric L -lactate dehydrogenases. J Biochem 2011, 150: 579–591. doi: 10.1093/jb/mvr100 10.1093/jb/mvr100
3. Baker PJ, Sawa Y, Shibata H, Sedelnikova SE, Rice DW: Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase. Nat Struct Biol 1998, 5: 561–567. doi: 10.1038/817 10.1038/817
4. Bernard N, Ferain T, Garmyn D, Hols P, Delcour J: Cloning of the D -lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli . FEBS Lett 1991, 290: 61–64. doi: 10.1016/0014–5793(91)81226-X 10.1016/0014-5793(91)81226-X
5. Buckel W, Barker HA: Two pathways of glutamate fermentation by anaerobic bacteria. J Bacteriol 1974, 117: 1248–1260.
Cited by
16 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献