Removal of a Methyl Group Causes Global Changes in ρ-Hydroxybenzoate Hydroxylase

ρ-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of ρ-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, 'in', 'out', and 'open' [Entsch, B., et al. (2005) 'Arch. Biochem. Biophys. 433', 297-311]. The open conformation is important for substrate binding and product release, the 'in' conformation for reaction with oxygen and hydroxylation, and the 'out' conformation for the reduction of FAD by NADPH. The 'open' conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the 'si' side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the 'in' conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the 'si' side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis.