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Biochemistry (Moscow)

, Volume 82, Issue 2, pp 176–185 | Cite as

Characterization of two recombinant 3-hexulose-6-phosphate synthases from the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z

  • O. N. Rozova
  • S. Y. But
  • V. N. KhmeleninaEmail author
  • A. S. Reshetnikov
  • I. I. Mustakhimov
  • Y. A. Trotsenko
Article

Abstract

Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His6, PHI-His6, and the two-domain proteinHPS–PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His6, HPS-His6 (2 × 20 kDa), and the fused protein HPS–PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose 5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the k cat/K m ratio, HPS-His6 had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS–PHI. AMP and ADP were powerful inhibitors of both HPS and HPS–PHI activities. The two-domain HPS–PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.

Keywords

methylotrophic bacteria Methylomicrobium alcaliphilum 20Z 3-hexulose-6-phosphate synthase 6-phospho-3 hexulose isomerase ribulose monophosphate cycle 

Abbreviations

FA

formaldehyde

F6P

fructose 6-phosphate

GPD

glucose-6-phosphate dehydrogenase

HPS

3-hexulose-6-phosphate synthase

LB

Luria–Bertani medium

ORF

open reading frame

PGI

phosphoglucoisomerase

PHI

6-phospho-3-hexulose isomerase

RuMP

ribulose monophosphate

Ru5P

ribulose 5-phosphate

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Copyright information

© Pleiades Publishing, Ltd. 2017

Authors and Affiliations

  • O. N. Rozova
    • 1
  • S. Y. But
    • 1
  • V. N. Khmelenina
    • 1
    Email author
  • A. S. Reshetnikov
    • 1
  • I. I. Mustakhimov
    • 2
  • Y. A. Trotsenko
    • 1
    • 2
  1. 1.Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of MicroorganismsRussian Academy of SciencesPushchino, Moscow RegionRussia
  2. 2.Pushchino State Institute of Natural SciencesPushchino, Moscow RegionRussia

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