Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli.

Autor: Reshetnikov AS; Laboratory of Methylotrophy, Federal Research Center, 'Pushchino Scientific Center for Biological Research, Russian Academy of Sciences' IBPM RAS, Pushchino, Moscow Region, Russia, 142290., But SY; Laboratory of Methylotrophy, Federal Research Center, 'Pushchino Scientific Center for Biological Research, Russian Academy of Sciences' IBPM RAS, Pushchino, Moscow Region, Russia, 142290., Rozova ON; Laboratory of Methylotrophy, Federal Research Center, 'Pushchino Scientific Center for Biological Research, Russian Academy of Sciences' IBPM RAS, Pushchino, Moscow Region, Russia, 142290., Mustakhimov II; Laboratory of Methylotrophy, Federal Research Center, 'Pushchino Scientific Center for Biological Research, Russian Academy of Sciences' IBPM RAS, Pushchino, Moscow Region, Russia, 142290., Khmelenina VN; Laboratory of Methylotrophy, Federal Research Center, 'Pushchino Scientific Center for Biological Research, Russian Academy of Sciences' IBPM RAS, Pushchino, Moscow Region, Russia, 142290. khmelenina@ibpm.pushchino.ru.
Jazyk: angličtina
Zdroj: Biotechnology letters [Biotechnol Lett] 2021 Jul; Vol. 43 (7), pp. 1421-1427. Date of Electronic Publication: 2021 Apr 16.
DOI: 10.1007/s10529-021-03130-0
Abstrakt: 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 His 6 -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.
Databáze: MEDLINE
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