Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction

Chiang, Chang-Hwa; Wymore, Troy; Rodríguez Benítez, Attabey; Hussain, Azam; Smith, Janet L.; Brooks, Charles L.; Narayan, Alison R.  H.

doi:10.1073/pnas.2218248120

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction

Journal Article · Tue Apr 04 00:00:00 EDT 2023 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2218248120· OSTI ID:2422804

^[1]; ^[2]; Rodríguez Benítez, Attabey ^[3]; Hussain, Azam ^[4]; ^[5]; ^[6]; ^[7]

Department of Chemistry, University of Michigan, Ann Arbor, MI 48109; Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109
Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794; Department of Chemistry, Stony Brook University, Stony Brook, NY 11794
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109
Macromolecular Science and Engineering Program, University of Michigan, Ann Arbor, MI 48109
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109; Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109; Department of Biophysics, University of Michigan, Ann Arbor, MI 48109
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109; Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes’ innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

View Accepted Manuscript (DOE)

Research Organization:: USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2422804

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 15 Vol. 120; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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