Acetate kinase-an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z

Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His₆-tagged...

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Bibliographic Details
Published in:	Antonie van Leeuwenhoek Vol. 108; no. 4; pp. 965 - 974
Main Authors:	Rozova, Olga N, Khmelenina, Valentina N, Gavletdinova, Juliya Z, Mustakhimov, Ildar I, Trotsenko, Yuri A
Format:	Journal Article
Language:	English
Published:	Cham Springer International Publishing 01-10-2015 Springer Nature B.V
Subjects:	acetate kinase Acetate Kinase - chemistry Acetate Kinase - genetics Acetate Kinase - metabolism acetates Adenosine diphosphate adenosine triphosphate affinity chromatography Aldehyde-Lyases - metabolism ATP Bacteria Bacteriology Biomedical and Life Sciences catalytic activity Chromatography, Affinity Cloning, Molecular Coenzymes - analysis E coli Enzyme Stability Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Gene Expression Profiling genes glycolysis heterologous gene expression ions Kinases Kinetics Life Sciences magnesium manganese Medical Microbiology Metabolic Networks and Pathways - genetics methane methanotrophs Methylobacter alcaliphilus Methylococcaceae - enzymology Methylococcaceae - genetics Microbiology Original Paper pentose phosphate cycle phosphoketolase phosphorylation Plant Sciences Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sequence Homology Soil Science & Conservation Substrate Specificity Temperature Acetate kinase Methanotropic bacteria Phosphoketolase
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Summary:	Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His₆-tagged AcK were determined. The M. alcaliphilum AcK (2 × 45 kDa) catalyzed the reversible phosphorylation of acetate into acetyl phosphate and exhibited a dependence on Mg²⁺ or Mn²⁺ ions and strong specificity to ATP/ADP. The enzyme showed the maximal activity and high stability at 70 °C. AcK was 20-fold more active in the reaction of acetate synthesis compared to acetate phosphorylation and had a higher affinity to acetyl phosphate (K ₘ 0.11 mM) than to acetate (K ₘ 5.6 mM). The k cₐₜ /K ₘ ratios indicated that the enzyme had a remarkably high catalytic efficiency for acetate and ATP formation (k cₐₜ/K ₘ = 1.7 × 10⁶) compared to acetate phosphorylation (k cₐₜ/K ₘ = 2.5 × 10³). The ack gene of M. alcaliphilum 20Z was shown to be co-transcribed with the xfp gene encoding putative phosphoketolase. The Blast analysis revealed the ack and xfp genes in most genomes of the sequenced aerobic methanotrophs, as well as methylotrophic bacteria not growing on methane. The distribution and metabolic role of the postulated phosphoketolase shunted glycolytic pathway in aerobic C₁-utilizing bacteria is discussed.
Bibliography:	http://dx.doi.org/10.1007/s10482-015-0549-5 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-6072 1572-9699
DOI:	10.1007/s10482-015-0549-5