6GQI

Thermocrispum municipale cyclohexanone monooxygenase bound to hexanoic acid

Summary for 6GQI

• Entry DOI: 10.2210/pdb6gqi/pdb
• Related: 5m10
• Descriptor: Cyclohexanone Monooxygenase from Thermocrispum municipale, FLAVIN-ADENINE DINUCLEOTIDE, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ... (6 entities in total)
• Functional Keywords: substrate specificity, biocatalysis, protein engineering, flavoprotein
• Biological source: Thermocrispum municipale
• Total number of polymer chains: 2
• Total formula weight: 123855.78

Authors

Mattevi, A.,Gomez Castellanos, J.R. (deposition date: 2018-06-07, release date: 2018-12-12, Last modification date: 2024-01-17)

Primary citation

Furst, M.J.L.J.,Romero, E.,Gomez Castellanos, J.R.,Fraaije, M.W.,Mattevi, A.
Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme.
ACS Catal, 8:11648-11656, 2018

PubMed Abstract: Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone monooxygenase from , a flavin-dependent Baeyer-Villiger monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates.

PubMed: 30687578
DOI: 10.1021/acscatal.8b03793

Experimental method

X-RAY DIFFRACTION (2 Å)