Two “classical” Old Yellow Enzymes from Chryseobacterium sp. CA49: Broad substrate specificity of Chr-OYE1 and limited activity of Chr-OYE2

•Two new “classic” Old Yellow Enzymes were mined from genome data.•Chr-OYE1 catalyzed the reduction of 18 of the tested substrates.•Chr-OYE1 catalyzed the reduction of an ynone via a two-step sequential process.•Chr-OYE2 catalyzed 3 of the substrates with lower activity than Chr-OYE1.•Mutant Chr-OYE2 showed significantly enhanced activity and stereoselectivity.

Two putative Old Yellow Enzyme (OYE) homologues, Chr-OYE1 and Chr-OYE2, were identified from the genome of Chryseobacterium sp. CA49 as new members of the “classical” subfamily. Chr-OYE1 and Chr-OYE2 were most closely related to the SYE4 from Shewanella oneidensis and NerA from Agrobacterium radiobacter with 41% and 45% identity, respectively. Both enzymes were expressed in Escherichia coli in soluble form, but their catalytic abilities as ene-reductases were quite different. Among the 19 substrate tested, Chr-OYE1 could catalyze the reduction of 18 of them including an ynone with excellent stereoselectivity for several prochiral ones, and its specific activity was roughly 1100-fold high than Chr-OYE2, which only catalyzed 3 of the substrates. After restoring the conserved tyrosine, Chr-OYE2 remained the same substrate spectrum, but showed significantly enhanced activity and stereoselectivity.

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