Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z


Autoři: Olga N. Rozova aff001;  Ildar I. Mustakhimov aff001;  Sergei Y. But aff001;  Aleksandr S. Reshetnikov aff001;  Valentina N. Khmelenina aff001
Působiště autorů: Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia aff001
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225054

Souhrn

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 μM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed.

Klíčová slova:

Enzyme metabolism – Enzymes – Methane – Molecular mass – Phosphates – Pyruvate – Citric acid cycle – Dehydrogenases


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