7LO1

FAD-dependent monooxygenase AfoD from A. nidulans

Summary for 7LO1

• Entry DOI: 10.2210/pdb7lo1/pdb
• Descriptor: FAD-dependent monooxygenase afoD, FLAVIN-ADENINE DINUCLEOTIDE, SULFATE ION, ... (4 entities in total)
• Functional Keywords: afod, flavin-dependent monooxygenase, fdmo, fad, flavoprotein
• Biological source: Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) (Aspergillus nidulans)
• Total number of polymer chains: 2
• Total formula weight: 100036.50

Authors

Rodriguez Benitez, A.,Smith, J.L.,Narayan, A.R.H. (deposition date: 2021-02-08, release date: 2022-04-27, Last modification date: 2023-10-25)

Primary citation

Chiang, C.H.,Wymore, T.,Rodriguez Benitez, A.,Hussain, A.,Smith, J.L.,Brooks 3rd, C.L.,Narayan, A.R.H.
Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction.
Proc.Natl.Acad.Sci.USA, 120:e2218248120-e2218248120, 2023

PubMed Abstract: 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.

PubMed: 37014851
DOI: 10.1073/pnas.2218248120

Experimental method

X-RAY DIFFRACTION (2.09 Å)