Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

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Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein involved in degradation of aromatic compounds, and it has become a model for enzymes involved in the oxygenation of a substrate. The chemical and kinetic mechanisms of this enzyme are described and integrated with an outline of the structure of the protein from crystallographic analysis. The structure is unusual because there is no recognizable domain for the binding of NADPH involved in the reaction. Recently, mechanistic studies of site-directed mutants, combined with structural analyses, have provided some exciting discoveries about protein function. The substrate during catalysis is largely isolated from solvent in the active site, a necessary condition for successful product formation. The flavin ring structure moves substantially in the active site, probably to enable substrate and product exchange into this site and possibly to regulate the reduction of the flavin by NADPH. A chain of H-bonds can connect p-hydroxy-benzoate in the active site of the enzyme with the protein surface. This chain is responsible for the reversible formation of substrate phenolate anion observed in the active site and partly responsible for the reactivity of this substrate.

Hydroxylation of aromatic compounds in bacteria by single component flavoprotein hydroxylases has been studied extensively for 40 years (1-3). Recently, several research groups, including ours, reported that hydroxylation of aromatic compounds can be catalyzed by two-protein or multiprotein monooxygenases. These include p-hydroxyphenylacetate 3-hydroxylase (HPAH) 3 from Pseudomonas putida (4), Escherichia coli (5), andAcinetobacter baumannii (6), phenol hydroxylase (PheA) from both Bacillus stearothermophilus BR219 and Bacillus thermoglucosidasius A7 (7, 8), chlorophenol-4-monooxygenase from Burkholderia cepacia AC1100 (9), 2,4,6-trichlorophenol monooxygenase from Ralstonia eutropha JMP134 (10), pyrrole-2-carboxylate monooxygenase from Rhodococcus sp (11), styrene monooxygenase from Pseudomonas VLB120 (12, 13), and p-nitrophenol hydroxylase from Bacillus sphaericus (14). Most of these enzyme systems consist of reductase and monooxygenase components, where the reductase component provides reduced flavin for the monooxygenase component to use for hydroxylating the aromatic substrate. The number of enzymes known in this class continues to increase and many more hypothetical proteins derived from genome projects have also been identified (15). Hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA) by HPAH is especially interesting because the same reaction is carried out by at least three types of two-component enzymes. The first HPAH purified was from P. putida, and it was shown to have FAD tightly bound to the smaller protein, and the larger protein (at that time) was thought to be a coupling protein enabling hydroxylation (4, 16). A different HPAH system was later isolated from E. coli W, and studies have shown that the smaller component (HpaC) is a flavin reductase that generates reduced FAD to be transferred to the larger component (HpaB) to hydroxylate HPA (5, 17). A detailed analysis of the mechanism of the E. coli-type HPAH is now in progress using the homologue from P. aeruginosa (18). The oxygenase in this system exhibits complex dynamics in catalysis (19).Our group has isolated HPAH from A. baumannii and shown that the enzyme is quite different from the analogous HPAH enzymes from either P. putida or E. coli (6,15,20). The A. baumannii HPAH is a two protein enzyme system consisting of a smaller reductase component (C 1 ) and a larger oxygenase component (C 2 ) (6). Sequence and several catalytic properties indicate that both components are different from others in the two protein class of aromatic hydroxylases (15,20). Our recent investigations of the reaction mechanisms of C 1 have shown that HPA controls the reduction of C 1 -bound FMN by NADH by shifting the enzyme into a more active conformation (20). By contrast, HPA has no effect at all on the activity of the reductase from the E. coli-type HPAH from P. aeruginosa (18). The HPAH from P. putida (above) (16) requires fresh examination based upon our current knowledge. It is possible that this enzyme system operates in ...

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