pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Abstract

Flavin-dependent monooxygenases catalyze the incorporation of a single atom of molecular oxygen into organic substrates (1, 2). The enzymes have been classified into six classes according to their catalytic and structural properties. They have also been categorized into two major types according to their protein components: a single-component type, in which reduction of a flavin cofactor and oxygenation of an organic substrate occurs within the same single polypeptide chain; and a two-component type, in which each reaction occurs on separate proteins (1, 2). Single-component monooxygenases have been identified since the 1960s and have been found to be involved in the aerobic metabolism of aromatic and aliphatic compounds in various organisms (2-4). The well known prototype for single-component monooxygenases is p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens (5, 6). The first enzyme identified as a two-component monooxygenase was bacterial luciferase (7). Nevertheless, most of the two-component monooxygenases have been identified only during the past decade and have increasingly emerged as common enzymes in nature that are involved in many important reactions in various microorganisms (1,8). Reactions catalyzed by two-component monooxygenases include oxygenation and halogenation of organic compounds such as p-hydroxyphenylacetate (9), phenol (10), trichorophenol (11, 12), p-nitrophenol (13), styrene (14 -16), alkane sulfonate (17, 18), and EDTA (19). Two-component monooxygenases are also involved in oxygenation and halogenation reactions in the biosynthetic pathways of actinorhodin (ActVA) (20), angucyclin (21), enediyne (SgcC) (22), rebeccamycin (RebH) (23), pyrrolnitrin (PrnA) (24), violacein (25), kutzneride (26), and differentiation-inducing factor-1 (27).All flavin-dependent monooxygenases perform oxygenation through the participation of a reactive intermediate, C4a-hydroperoxy-flavin, which has been well documented and detected by transient kinetics for the reactions of singlecomponent monooxygenases. These monooxygenases include p-hydroxybenzoate hydroxylase (PHBH) 3 (5, 6), phenol hydroxylase (28), melilotate hydroxylase (29), antranilate hydroxylase (30), Baeyer-Villiger monooxygenases (31, 32), 2-methyl-3-hydroxypyridine-5-carboxylic oxygenase * This work was supported by Grants BRG5180002 (to P. C.) and MRG5380240 (to J. S.) from the Thailand Research Fund and a grant from the Faculty of Science, Mahidol University (to P. C.

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

“…1-5. All His-120 variants show similar kinetic patterns as those of H120K and wild-type enzymes (15), and the reactions are depicted in Fig. 3.…”

“…The spectrum in the inset in Fig. 2A had a maximum absorption peak at 390 nm, similar to that of a C4a-hydroperoxy-FMN intermediate in the reactions of wild-type C 2 and other variants (15,21,27). Therefore, the first phase shown in Fig.…”

“…High purity FMN was produced by converting FAD to FMN using snake venom from Crotalus adamanteus (28) according to the protocol described in Sucharitakul et al (22). C 1 , wild-type C 2 , and C 2 variants were expressed and purified as previously described (15)(16)(17). The concentrations of the following compounds were determined using the known extinction coefficients at pH 7. enzyme solution was reduced with an equal reducing equivalent of sodium dithionite (a solution of 5 mg/ml in 100 mM potassium phosphate buffer pH 7.0).…”

“…The enzyme from Acinetobacter baumannii is composed of a reductase component (C 1 ) and an oxygenase component (C 2 ), and it catalyzes the hydroxylation of HPA to 3,4-dihydroxyphenylacetate (DHPA) (16,17). 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). Although the ability of C 2 to prolong the lifetime of C4a-hydroperoxy flavin is necessary for substrate hydroxylation (27), the enzyme must have another fine-tuned mechanism for efficient oxygen transfer because between pH 6.0 and 10.0 the product conversion percentage is ϳ90% without wasteful H 2 O 2 production, and the rate constants for hydroxylation remain constant at 15-17 s Ϫ1 (15). The pH independence of the C 2 reaction implies that the protonation status of key components required for oxygen atom transfer is unchanged in this pH range.…”