Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii

Abstract

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 ...

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

“…A corresponding tryptophan is conserved with similar orientation in medium-chain acyl-CoA dehydrogenase and nitroalkane oxidase (13,14) and may represent a signature residue for the flavoenzymes sharing this folding topology. C 2 binds FMNH Ϫ more tightly than oxidized FMN with a 200-fold difference in K d (7). Consistently, no FMN bound to crystals has been observed when oxidized FMN was used in cocrystallizations.…”

“…4). With such an arrangement, the additional AMP moiety of FAD would easily hang out in the solvent or rest on the tetramer surface; this observation explains why both FMNH Ϫ and FADH Ϫ work equally well in the hydroxylation reaction (7,9).…”

“…In the absence of HPA, molecular oxygen may have easier access to the cavity by exploiting the inherent flexibility of Phe-266 side chain. This sort of protection exerted by HPA may also account for the stability of the dead-end complex that forms when HPA binds to the C 2 :C4a-hydroxyflavin (7).…”

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

“…6-OH-FAD was a gift from David Ballou (University of Michigan). 6-OH FMN was generated from 6-OH-FAD using snake venom phosphodiesterase I and then purified as described (51) and the conversion and purity was checked in HPLC. Detail has been described in SI Materials and Methods.…”

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

“…In the specific case of the DKCMOs, both isoenzymes have been reported to exploit separate copies of the same monomeric 36 kDa flavinreducing component referred to as 'NADH dehydrogenase' . A unique feature of the DKCMOs from camphor-grown NCIMB 10007 that distinguishes them from all other TCMOs characterized to date, including other class C flavin monooxygenases such as various bacterial luciferases (Meighen, 1991), class D flavoprotein monooxygenases such as 4-hydroxyphenylacetate-3-monooxygenase from E. coli W (Prieto et al, 1996;Galán et al, 2000) and Acinetobacter baumanii (Chaiyen et al, 2001;Sucharitakul et al, 2006), and styrene monooxygenases from various Pseudomonas and Rhodococcus species (O'Leary et al, 2002) concerns the extent to which the participating components can reasonably be expected to exhibit functional dependence on each other. The relevant genes encoding both the biooxygenating and the reductase components of these previously studied multicomponent monooxygenases are located in the same operon or proximal DNA, and hence are likely to have some evolved dedicated functional interdependence.…”