Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Abstract

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ 3 -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

“…Structurally, the position of this arginine relative to the cofactor, corresponding to R209 in CHMO, is found to be well conserved. The T218A mutation in PAMO showed little effect on catalytic efficiency (20), consistent with a relatively minor role for T217 of OTEMO in NADPH binding.…”

“…The importance of R216 for NADPH binding and the specificity for NADPH over NADH is reflected in the large decrease in the k cat /K m value, resulting from a 3 orders of magnitude increase in the K m observed for the corresponding R217A or R217L mutant PAMO enzymes from T. fusca (20). The corresponding residue in HAPMO, R339, is also critical for NADP ϩ recognition (28).…”

Snapshots of Enzymatic Baeyer-Villiger Catalysis

“…This observation is consistent with multisubstrate kinetic studies. The order of NADPH and substrate binding was studied for the R217A mutant of PAMO, which features a K m for NADPH (320 M) suited for this type of experiments (the mutation is unlikely to affect the reaction mechanism because Arg-217 is located far away from the flavin) (39). When the observed reaction rates were plotted according to LineweaverBurk method, the characteristic pattern of parallel lines was observed, indicating an ordered binding of substrates.…”

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

“…In the presence of D-Lys the k cat /K NADPH value was ϳ4-fold higher. Thus, NbtG does not have significant coen- zyme selectivity, in contrast to other Class B flavin monooxygenases, which are all highly selective for NADPH (1,(37)(38)(39). Lysine Hydroxylation-NbtG displayed significant activity as measured by the oxygen consumption assay.…”