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Microorganisms 2015, 3(1), 47-59; doi:10.3390/microorganisms3010047

Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b

1
Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Prospect Nauki 5, Pushchino 142290, Russia
2
Department of Microbiology and Biotechnology, Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino 142290, Russia
*
Author to whom correspondence should be addressed.
Academic Editors: Marina G. Kalyuzhnaya and Ludmila Chistoserdova
Received: 30 December 2014 / Revised: 21 January 2015 / Accepted: 5 February 2015 / Published: 27 February 2015
(This article belongs to the Special Issue Microbial C1 Metabolism)
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Abstract

We have expressed the l-malate dehydrogenase (MDH) genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa) enzyme displaying nearly equal reductive (malate formation) and oxidative (oxaloacetate formation) activities and higher affinity to malate (Km = 0.11 mM) than to oxaloacetate (Km = 0.34 mM). The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa), two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM) than to malate (Km = 1.28 mM). The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed. View Full-Text
Keywords: l-malate dehydrogenase; catalytic efficiency; thermostability; tricarboxylic acid cycle; methanotrophs; Methylomicrobium alcaliphilum; Methylosinus trichosporium l-malate dehydrogenase; catalytic efficiency; thermostability; tricarboxylic acid cycle; methanotrophs; Methylomicrobium alcaliphilum; Methylosinus trichosporium
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Rozova, O.N.; Khmelenina, V.N.; Bocharova, K.A.; Mustakhimov, I.I.; Trotsenko, Y.A. Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b. Microorganisms 2015, 3, 47-59.

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