Crystal Structure of the Oxygenase Component (HpaB) of the 4-Hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8

Abstract

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 ␤5 and ␤6 and of the loop between ␤8 and ␤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 C4a-hydroperoxyflavin intermediate. The 4-hydroxyphenylacetate (4HPA)2 3-monooxygenase catalyzes hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate (DHPA). This enzyme is involved in the initial step of the degradation pathway of 4HPA. It uses molecular oxygen and reduced flavin for hydroxylation and NAD(P)H for flavin reduction. Thus, the enzymatic reaction is separated into two steps, the reduction of flavin to generate two reducing equivalents using NAD(P)H as an electron donor and the hydroxylation of substrates using molecular oxygen and reduced flavin. This enzyme has been isolated from Escherichia coli (1-4), Pseudomonas putida (5, 6), Klebsiella pneumoniae (7), and Acinetobacter baumannii (8, 9). The small component of these enzymes is the flavin reductase, which generates reduced flavin for the oxygenase component to oxygenate substrates. Hydroxylation activity is dependent on the presence of the reductase component (1-9). Galán et al. (10) have classified these enzymes into the two-component flavin-diffusible monooxygenase (TC-FDM) family (10). The TC-FDM family includes monooxygenases targeting various substrates such as 4HPA, chlorophenol (11), styrene (12), phenol (13), p-nitrophenol (14), nitrilotriacetate (15), EDTA (16), and aliphatic sulfonate (17). Amino acid sequences of reductase components are relatively similar to each other, whereas those of oxygenase components differ, which may reflect substrate specificity for either aromatic or non-aromatic compounds (14). Recent studies indicate that members of the TC-FDM family that act on aromatic compounds can be further divided into two groups. One group includes an enzyme from A. baumannii (and probably one from P. putida), and the other includes the remaining members (the E. coli enzyme is representative of this group). The reductase component (C 1 ) from A. baumannii is larger than E. coli-type reductases of the TC-FDM family and is stimulated by the presence of the substrate (18). In contrast, E. colitype reductases are not affected by ...

Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

“…Ser-146 interactions with the substrate are not mandatory for hydroxylation but assist in positioning the enzyme-bound HPA in an optimum geometry for hydroxylation. According to x-ray structures of two-component monooxygenases that utilize phenolic compounds as substrates, these two residues are well conserved (24,31,32). Our findings imply that the conserved His/Ser pairs found in other two-component monooxygenases may have similar functional roles.…”

“…Homologues of these residues are found in the oxygenase component (HpaB) of HPAH from T. thermophilus HB8, in which the O atom of Thr-104 and the N ⑀ of His-142 are 2.5 and 2.8 Å from the hydroxyl group of HPA, respectively (24), and in the oxygenase involved in the cholesterol catabolic pathway of Mycobacterium tuberculosis, in which His-92 and Ser-118 are about 2.4 Å from the hydroxyl group of the modeled 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione substrate (32). For the oxygenase (TftD) of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100, His-289 was proposed to interact with a hydroxyl group of 2,5-dichlorohydroquinone docked in the structure of TftD (31).…”

“…Kinetic studies of HPAHs from Pseudomonas putida (18), Pseudomonas aeruginosa (19), Escherichia coli W (20), and A. baumannii (8,15,21,22) have been reported. X-ray structures of the oxygenase components from A. baumannii at 2.3-2.8 Å resolution (23) and Thermus thermophilus HB8 at 1.3-2.0 Å resolution (24,25) and the reductase component from Sulfolobus tokodaii (26) at 1.7-2.3 Å resolution have been solved. Recent site-directed mutagenesis studies of the HPAH from A. baumannii have shown that Ser-171 is a key residue for stabilization of the reactive intermediate, C4a-hydroperoxy flavin, whereas His-396 facilitates intermediate formation (27).…”

Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression

“…These differences in gene regulation and substrate specificity suggest that the 4-NP hydroxylase system might have been completely ramified for 4-NP oxidation from 4-HPA 3-hydroxylase systems, although both hydroxylase systems are expected to share the same origin, judging from their amino acid sequence identities. Very recently, the crystal structure of a different 4-HPA 3-monooxygenase (HpaB) of Thermus thermophilus HB8 was determined and its catalytic mechanism was discussed (18). To understand the catalytic mechanism of our enzyme, we are also trying to elucidate the structure through crystallographic study and X-ray analysis.…”

The C-terminal Domain of 4-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii Is an Autoinhibitory Domain

“…HPAH belongs to a large family of two-component flavin-dependent monooxygenases. Catalytic and structural studies of the enzymes from several bacterial species, including Pseudomonas putida (16,17), Pseudomonas aeruginosa (15), Escherichia coli (18), Klebsiella pneumonia (19), Sulfolobus tokodaii (20), Thermus thermophilus (21,22), and A. baumannii (12,(23)(24)(25)(26)(27)(28)(29)(30)(31), have been carried out. HPAH consists of a reductase component (C 1 ) that catalyzes reduction of FMN to generate FMNH Ϫ for its oxygenase (C 2 ), which catalyzes the oxygenation of HPA (12,23).…”