Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope

Enzyme Microb Technol. 2018 Jun:113:1-8. doi: 10.1016/j.enzmictec.2018.02.005. Epub 2018 Feb 17.

Abstract

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering.

Keywords: Biocatalysis; C-H activation; CYP116B; Cytochrome P450 monooxygenase; Demethylation; Halomonas sp.

MeSH terms

  • Biocatalysis
  • Cloning, Molecular / methods*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Demethylation
  • Gene Expression Regulation, Enzymologic*
  • Halomonas / enzymology*
  • Halomonas / genetics
  • Hydroxylation
  • NADP / metabolism*
  • Phylogeny
  • Substrate Specificity
  • Sulfates / chemistry

Substances

  • Sulfates
  • NADP
  • Cytochrome P-450 Enzyme System