Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction.
A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP+and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically-modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center that restrains the flavin-peroxide intermediate in a catalytically-competent orientation. Strikingly, the regio- and stereoselectivities of the reaction are essentially unaffected by cofactor modifications. These observations indicate a remarkable robustness of this complex multi-cofactor active site, which has implications for enzyme design based on cofactor engineering approaches.
Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy.
Christian
C
Martinoli
Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Hanna M
HM
Dudek
Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy.
Roberto
R
Orru
Departments of Biochemistry and Chemistry, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA.
Dale E
DE
Edmondson
Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Marco W
MW
Fraaije
Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy.
Andrea
A
Mattevi
biocatalysis; cofactors; flavin; monooxygenation; nicotinamide