Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family

Martin Marek, Radka Chaloupkova, Tatyana Prudnikova, Yukari Sato, Pavlina Rezacova, Yuji Nagata, Ivana Kuta Smatanova, Jiri Damborsky

Research output: Contribution to journalArticlepeer-review

Abstract

Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and α4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (ΔTm = −5 to 6 °C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear β-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.

Original languageEnglish
Pages (from-to)1352-1362
Number of pages11
JournalComputational and Structural Biotechnology Journal
Volume18
DOIs
Publication statusPublished - 2020

Keywords

  • Access tunnel
  • Biocatalysis
  • Enantioselectivity
  • Enzyme engineering
  • Haloalkane dehalogenase (HLD)
  • Loop-helix transplantation
  • Protein design
  • X-ray crystallography

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Structural Biology
  • Biochemistry
  • Genetics
  • Computer Science Applications

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