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Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants.

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Walton AZ, Sullivan B, Patterson-Orazem AC, Stewart JD
OpenAccess logo ACS Catal 4 2307-2318 (2014)
Cited: 15 times
EuropePMC logo PMID: 25068071

Abstract

A systematic saturation mutagenesis campaign was carried out on an alkene reductase from Pichia stipitis (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis-Hillman adducts to the corresponding S products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high R selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee (R)). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations.

Articles - 4m5p mentioned but not cited (1)

  1. Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants. Walton AZ, Sullivan B, Patterson-Orazem AC, Stewart JD. ACS Catal 4 2307-2318 (2014)


Reviews citing this publication (4)

  1. Biocatalytic Reduction Reactions from a Chemist's Perspective. Hollmann F, Opperman DJ, Paul CE. Angew Chem Int Ed Engl 60 5644-5665 (2021)
  2. Applications of protein engineering to members of the old yellow enzyme family. Amato ED, Stewart JD. Biotechnol Adv 33 624-631 (2015)
  3. Old yellow enzymes: structures and structure-guided engineering for stereocomplementary bioreduction. Shi Q, Wang H, Liu J, Li S, Guo J, Li H, Jia X, Huo H, Zheng Z, You S, Qin B. Appl Microbiol Biotechnol 104 8155-8170 (2020)
  4. "A Study in Yellow": Investigations in the Stereoselectivity of Ene-Reductases. Parmeggiani F, Brenna E, Colombo D, Gatti FG, Tentori F, Tessaro D. Chembiochem 23 e202100445 (2022)

Articles citing this publication (10)

  1. Discovery, Characterisation, Engineering and Applications of Ene Reductases for Industrial Biocatalysis. Toogood HS, Scrutton NS. ACS Catal 8 3532-3549 (2019)
  2. Gram-Scale Synthesis of Chiral Cyclopropane-Containing Drugs and Drug Precursors with Engineered Myoglobin Catalysts Featuring Complementary Stereoselectivity. Bajaj P, Sreenilayam G, Tyagi V, Fasan R. Angew Chem Int Ed Engl 55 16110-16114 (2016)
  3. Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution. Sun Z, Lonsdale R, Kong XD, Xu JH, Zhou J, Reetz MT. Angew Chem Int Ed Engl 54 12410-12415 (2015)
  4. Alkene hydrogenation activity of enoate reductases for an environmentally benign biosynthesis of adipic acid. Joo JC, Khusnutdinova AN, Flick R, Kim T, Bornscheuer UT, Yakunin AF, Mahadevan R. Chem Sci 8 1406-1413 (2017)
  5. Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues. Nett N, Duewel S, Richter AA, Hoebenreich S. Chembiochem 18 685-691 (2017)
  6. Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal. Ying X, Yu S, Huang M, Wei R, Meng S, Cheng F, Yu M, Ying M, Zhao M, Wang Z. Molecules 24 E1057 (2019)
  7. Stereoselectivity Switch in the Reduction of α-Alkyl-β-Arylenones by Structure-Guided Designed Variants of the Ene Reductase OYE1. Crotti M, Parmeggiani F, Ferrandi EE, Gatti FG, Sacchetti A, Riva S, Brenna E, Monti D. Front Bioeng Biotechnol 7 89 (2019)
  8. Catalytic Performance of a Class III Old Yellow Enzyme and Its Cysteine Variants. Scholtissek A, Gädke E, Paul CE, Westphal AH, van Berkel WJH, Tischler D. Front Microbiol 9 2410 (2018)
  9. Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral. Wang T, Wei R, Feng Y, Jin L, Jia Y, Yang D, Liang Z, Han M, Li X, Lu C, Ying X. Molecules 26 5040 (2021)
  10. Engineered Myoglobin Catalysts for Asymmetric Intermolecular Cyclopropanation Reactions. Siriboe MG, Fasan R. Bull Jpn Soc Coord Chem 80 4-13 (2022)


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