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 AcK were determined. The M. alcaliphilum AcK (2 × 45 kDa) catalyzed the reversible phosphorylation of acetate into acetyl phosphate and exhibited a dependence on Mg(2+) or Mn(2+) 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 × 10(6)) compared to acetate phosphorylation (k cat/K m = 2.5 × 10(3)). 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.
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, 142290, Russia, rozovaolga1@rambler.ru.
Olga N
ON
Rozova
Valentina N
VN
Khmelenina
Juliya Z
JZ
Gavletdinova
Ildar I
II
Mustakhimov
Yuri A
YA
Trotsenko
D000084:Acetate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008772:Methylococcaceae / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity; D013696:Temperature
D003067:Coenzymes; D011994:Recombinant Proteins; D000084:Acetate Kinase; D000446:Aldehyde-Lyases; C022367:phosphoketolase