Crystal Structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8

The 4-hydroxyphenylacetate ( 4HPA) 3-monooxygenase is involved in the initial step of the 4HPA degradation pathway and catalyzes 4HPA hydroxylation to 3,4-dihydroxyphenylacetate. This enzyme consists of two components, an oxygenase ( HpaB) and a reductase ( HpaC). To understand the structural basis of the catalytic mechanism of HpaB, crystal structures of HpaB from Thermus thermophilus HB8 were determined in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4HPA. Structural analysis revealed that the binding and dissociation of flavin are accompanied by conformational changes of the loop between beta 5 and beta 6 and of the loop between beta 8 and beta 9, leading to preformation of part of the substrate-binding site ( Ser-197 and Thr-198). The latter loop further changes its conformation upon binding of 4HPA and obstructs the active site from the bulk solvent. Arg-100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4-ahydroperoxyflavin intermediate.