Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from <i>Gluconobacter oxydans</i>

Yin, Biaolin; Deng, Jiao; Lim, Lirong; Yuan, Yunbin; Wei, Dongzhi

doi:10.1080/09168451.2014.993355

Cited by 2 publications

(3 citation statements)

References 47 publications

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“…In contrast to the substrate binding mode of the wild type enzyme, the W116F mutation enabled the substrate to bind with a flipped orientation in the active site, and thus reverse the enantioselectivity, while maintaining the same mechanism of trans-hydrogenation of C=C bond [23,24]. W116 is not the sole determinant of enantioselectivity in OYEs, and the enantioselectivity switches seemed to vary by enzyme: Y78, I113, and F247 in OYE2.6 [25]; C26, I69, and H167 in ene reductases YqjM [26]; and W66 and W100 in OYE from Gluconobacter oxydans (Gox0502) [27]. The study of enantioselectivity alteration in OYEs rarely use citral as substrate.…”

Section: Introductionmentioning

confidence: 99%

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Ying

Huang

et al. 2019

Molecules

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The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal—a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.

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Section: Introductionmentioning

confidence: 99%

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Ying

Huang

et al. 2019

Molecules

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“…values for the reduction of ( E / Z )-citral was lower than that for the reduction of ( E )-citral. To improve the ( R )-enantioselectivity in the reduction of ( E / Z )-citral, site-directed mutagenesis was conducted to test whether key residues critical for other OYEs determined the enantioselectivity of OYE3 [ 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. OYE3 and its 12 variants were expressed in Escherichia coli and purified for the evaluation of catalytic performance ( Figure S1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Protein engineering has been proved to be powerful for overcoming the deficiencies of native OYEs, and successful examples of the improvement of enantioselectivity are accumulating [ 16 , 17 , 18 , 19 ]. Site-directed mutagenesis technique was widely used for the modification of OYEs, and various key residues determining the enantioselectivity were reported, including W116 and F296 in OYE1 [ 20 , 21 , 22 ], Y78, I113, and F247 in OYE2.6 [ 23 ]; C26, I69, and H167 in ene reductases YqjM [ 24 ]; W100 in OYE from Gluconobacter oxydans (Gox0502); and W66 in NCR ene reductase [ 25 , 26 ]. The common strategy engineering the enantioselectivity of OYEs in citral reduction was to change the substrate-binding mode, enabling one citral isomer to bind with a flipped orientation, while maintaining the orientation of the other citral isomer similar to the wild-type enzyme [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

et al. 2021

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The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

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Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacter oxydans

Abstract: (2015) Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacteroxydans, Bioscience, Biotechnology, and Biochemistry, 79:3,[410][411][412][413][414][415][416][417][418][419][420][421]

Cited by 2 publications

References 47 publications

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

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