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.
Highlights
The malate dehydrogenase (MDH, L-malate: NAD oxidoreductase, EC 1.1.1.37) catalyzing the NAD(P)+/NAD(P)H-dependent interconversion of L-malate to oxaloacetic acid (OAA) is widespread in the three domains of life
M. trichosporium OB3b were successfully expressed in E. coli BL21 (DE3), and the His6-tagged proteins were purified from crude extracts of E. coli cells by single-step metal-chelating affinity chromatography
(70 kDa) implies that the enzyme exists as a homodimer, whereas the Mr of the M. trichosporium MDH
Summary
The malate dehydrogenase (MDH, L-malate: NAD oxidoreductase, EC 1.1.1.37) catalyzing the NAD(P)+/NAD(P)H-dependent interconversion of L-malate to oxaloacetic acid (OAA) is widespread in the three domains of life It plays crucial roles in many metabolic pathways, including the tricarboxylic acid (TCA) cycle, energy generation and the formation of metabolites for biosynthesis. Aerobic bacteria utilizing methane as a sole source of carbon and energy (methanotrophs) belong to the Alpha and Gamma classes of Proteobacteria and the phylum Verrucomicrobia [1,2] Since these bacteria are able to obtain energy from the oxidation of reduced C1 compounds, the TCA cycle would not be an obligatory way for energy generation. The gammaproteobacterial methanotrophs, such as Methylomicrobium alcaliphilum 20Z, use the ribulose monophosphate (RuMP)
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