Abstract
Objectives
Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments.
Results
Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as His6-tagged proteins were characterized. The simultaneous substitutions of arginine-180, arginine-198 and serine-178 residues by alanine in the enzyme pocket sequence as well as other amino acid changes decreased both NADPH- and NADH-dependent activities in comparison to the wild-type enzyme. The replacement of serine-156 by glutamic acid decreased NADPH-dependent activity at least 7000-fold but NADH-dependent activity only by threefold. The replacement of serine-156 by aspartic acid decreased NADPH-dependent activity 70-fold with fair preservation of activity and specificity to NADH.
Conclusions
These results demonstrated a relevance of Asp156 in the interaction of AcuI from E. coli K-12 with NADH as a coenzyme. These findings may provide reference information for shifting coenzyme specificity of acrylyl-CoA reductases.
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Acknowledgements
This work was supported by Russian Foundation for Basic Research, project 18-29-07044 MK
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ASR conceived and designed the experiments, analyzed, collected and interpreted all the data obtained by other authors, obtained point mutations of the acuI gene. SYB determined the activity of mutant forms of the AcuI enzyme. IIM performed the part of the work related to the synthesis and chromatographic purification of acrylyl CoA. ONR performed the part of the work related to modeling the tertiary structure of the mutated AcuI enzyme. VNK is responsible for all parts of the work, resolving issues related to the accuracy and integrity of the work. All authors take part in the final approval of the version of the article for publication.
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Reshetnikov, A.S., But, S.Y., Rozova, O.N. et al. Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli. Biotechnol Lett 43, 1421–1427 (2021). https://doi.org/10.1007/s10529-021-03130-0
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DOI: https://doi.org/10.1007/s10529-021-03130-0