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Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations†

Engineering catalytic properties and thermal stability of plant formate dehydrogenase by... The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased KмHCOO− from 1.5 to 4.1–5.0 mM, whereas the KмNAD+ values remained almost unchanged in the range from 9.1 to 14.0 μM, which is close to wt-SoyFDH (13.3 μM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s−1, respectively; while that of the wild-type enzyme was 2.9 s−1. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the Tm values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Engineering, Design and Selection Oxford University Press

Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations†

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References (22)

Publisher
Oxford University Press
Copyright
© The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected]
Subject
Original articles
ISSN
1741-0126
eISSN
1741-0134
DOI
10.1093/protein/gzs084
pmid
23100543
Publisher site
See Article on Publisher Site

Abstract

The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased KмHCOO− from 1.5 to 4.1–5.0 mM, whereas the KмNAD+ values remained almost unchanged in the range from 9.1 to 14.0 μM, which is close to wt-SoyFDH (13.3 μM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s−1, respectively; while that of the wild-type enzyme was 2.9 s−1. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the Tm values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.

Journal

Protein Engineering, Design and Selection – Oxford University Press

Published: Nov 24, 2012

Keywords: modeling rational design site-directed mutagenesis soya Glycine max structure

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