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

Abstract

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.

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach

“…Both the growth rate and the increase in acetate excretion have been previously observed as main outcomes of O 2 -limiting growth 16 , 40 . Furthermore, the kinetic properties of purified acetate kinase from M. alcaliphilum 20Z support the in-silico prediction (Table 3 31 ).…”

Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

“…PKT orthologs have been observed in the genomes of an array of methanotrophic and methylotrophic bacteria 16 ; however, their role in CH 4 metabolism remains elusive. M. buryatense encodes two putative PKT isoforms, PktA and PktB, that share 63.7% amino acid identity ( Figure S1).…”

“…PKTs have been characterized in obligate and heterofermentative bacteria and some species of fungi where they enable an alternative pathway for sugar-phosphate degradation. Interestingly, PKT orthologs have been observed in the genomes of an array of C1-utilizing microbes 16 , and it has been postulated that the PKT pathway may serve an important metabolic role in methanotrophic, methylotrophic, and autotrophic bacteria 16,17 .…”

Ectoine degradation pathway in halotolerant methylotrophs

“…alcaliphilum is an obligate C1-utilizer incapable of using any organic compound as a single growth substrate; nevertheless, acetate and aspartate, the products of the ectoine degradation route, can enter the central biochemical pathways in the methanotroph. Acetate can enter the tricarboxylic acid (TCA) cycle after the activation by acetate kinase [31]. In addition, the oxaloacetate derived from aspartate can be reduced to malate, which is subsequently converted to pyruvate via the malic enzyme [32].…”