Dissecting the low catalytic capability of flavin-dependent halogenases
Aisaraphon Phintha, Kridsadakorn Prakinee, A. Jaruwat, Narin Lawan, Surawit Visitsatthawong, Chadaporn Kantiwiriyawanitch, Warangkhana Songsungthong, Duangthip Trisrivirat, Pirom Chenprakhon, Adrian J. Mulholland, Karl‐Heinz van Pée, P. Chitnumsub, Pimchai Chaiyen
Abstract
reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.