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

• Published in: PloS one Vol. 14; no. 11; p. e0225054
• Main Authors: Rozova, Olga N, Mustakhimov, Ildar I, But, Sergei Y, Reshetnikov, Aleksandr S, Khmelenina, Valentina N
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.11.2019; Public Library of Science (PLoS)
• Subjects: Air pollution; Amino acids; Anthropogenic factors; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Biocatalysts; Biochemistry; Bioconversion; Biological research; Biology and Life Sciences; Biosphere; Biotechnology; Carbon; Chelation; Chromatography; Cloning; Cloning, Molecular; Decarboxylation; Dehydrogenases; Disruption; E coli; Earth Sciences; Ecology and Environmental Sciences; Electrophoresis; Energy Metabolism; Enzymes; Escherichia coli; Gel electrophoresis; Gel filtration; Gene Expression; Genetic research; Genomes; Genomic analysis; Genomics - methods; Greenhouse effect; Greenhouse gases; Intracellular; Mae gene; Malate; Malic enzyme; Metabolic Networks and Pathways; Metabolism; Metals - metabolism; Methane; Methane - metabolism; Methanotrophic bacteria; Methylococcaceae - classification; Methylococcaceae - enzymology; Methylococcaceae - genetics; Methylococcaceae - metabolism; Microorganisms; Mutagenesis; Mutation; NAD; Natural gas; Phenotype; Phosphates; Phylogeny; Physical Sciences; Physiological aspects; Physiology; Pyruvic acid; Recombinant Proteins; Salinity tolerance; Substrates; Tetracyclines; Russia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0225054

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.