Role of NAD⁺-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b.
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.
Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Prospect Nauki 5, Pushchino 142290, Russia. olgan.rozova@gmail.com.
Olga N
ON
Rozova
Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Prospect Nauki 5, Pushchino 142290, Russia. khmelenina@rambler.ru.
Valentina N
VN
Khmelenina
Department of Microbiology and Biotechnology, Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino 142290, Russia. ksenia.alpha4160@mail.ru.
Ksenia A
KA
Bocharova
Department of Microbiology and Biotechnology, Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino 142290, Russia. mii80@rambler.ru.
Ildar I
II
Mustakhimov
Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Prospect Nauki 5, Pushchino 142290, Russia. trotsenko@ibpm.pushchino.ru.|Department of Microbiology and Biotechnology, Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino 142290, Russia. trotsenko@ibpm.pushchino.ru.
Yuri A
YA
Trotsenko
Methylomicrobium alcaliphilum; Methylosinus trichosporium; catalytic efficiency; l-malate dehydrogenase; methanotrophs; thermostability; tricarboxylic acid cycle
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