Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 188, Issue 2, Pages 93-101Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2014.10.003
Keywords
Crystal structure; Nitrilase; Syechocystis sp PCC6803; Substrate specificity determination
Funding
- National University of Singapore (Suzhou) Research Institute [R-2012-N-007]
- Singapore Ministry of Education [R-154-000-618-112]
- Fundamental Research Funds for the Central Universities, '973' Program [2012CB721003]
- National Natural Science Foundation [21276084/B060804]
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Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys-Glu-Lys catalytic triad to hydrolyze non-peptide carbon-nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 angstrom resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates. (C) 2014 Elsevier Inc. All rights reserved.
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