An Aromatic Hydroxylation Reaction Catalyzed by a Two-component FMN-dependent Monooxygenase

Abstract

The ActVA-ActVB system from Streptomyces coelicolor is a twocomponent flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.The two-component flavin-dependent monooxygenases have recently emerged as an important class of enzyme systems involved in biological oxidations (1). They are composed of two enzymes. First, a NAD(P)H:flavin oxidoreductase that catalyzes the reduction of free flavin, flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), by reduced pyridine nucleotides, NADPH or NADH (2-7). Second, an oxygenase that binds the resulting free reduced flavin together with the substrate in the active site (8 -20). There is general agreement that the reaction proceeds through an oxidation of the flavin by molecular oxygen to generate a flavin hydroperoxide intermediate (21). This is followed by transfer of a single oxygen atom from the electrophilic peroxide to the substrate, generating the oxidized product of the reaction (21). Therefore in this two-component system, NAD(P)H oxidation and the hydroxylation reaction are catalyzed by separate polypeptides. In all cases, with the exception of luciferase (22), there is no evidence for an interaction between the two components and a channeling mechanism allowing the flavin to travel from one protein to another within a protein complex. Accordingly, in most cases, the oxidoreductase component can be replaced by other flavin reductases, including the non-homologous ones from other organisms (4, 5). The flavin transfer process from the oxidoreductase to the oxygenase is thus simply under thermodynamic control (5, 20).The most extensively studied oxygenase components so far are those utilizing FADH 2 as a cofactor, such as 4-hydroxyphenylacetate monooxygenase (HpaB) from Escherichia coli (8), phenol hydroxylase (PheA1) from Bacillus thermoglucosidaflurescens (4), and styrene monooxygenase (StyA) from Pseudomonas fluorescens (12, 13). In contrast, the oxygenases utilizing FMNH 2 (termed FMN red herein) have been much less investigated. Examples are tho...

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

“…These data, which are consistent with our previous results from experiments at 25°C, clearly show that ActVA strongly stabilizes the C(4a)-FMN-OOH intermediate (17,24 Fig. 2, the reported data were observed with a final O 2 concentration of 595 M. The reaction was followed at: (i) 365 nm, a wavelength that is useful for monitoring the formation of the C(4a)-FMN-OOH intermediate species (22,29,30) and at which DHK red contributes to the absorbance minimally; (ii) 445 nm to monitor the formation of FMN ox ; and (iii) 520 nm to monitor the formation of the hydroxylated DHK ox product (DHK ox -OH) (24). In addition, fluorescence emission at 530 nm (excitation at 390 nm) was recorded to follow the formation of C(4a)-FMN-OH (hydroxy-FMN) and FMN ox species.…”

“…In the absence of the substrate, the C(4a)-FMN-OOH intermediate forms rapidly (ϳ3 ϫ 10 4 M Ϫ1 s Ϫ1 ) and is remarkably stable, as previously observed (17,24), and thus slowly converts to FMN ox (and probably H 2 O 2 ) with a half-life of ϳ1400 s. Formation of the hydroperoxide intermediate in the absence of substrate has also been observed for other members of the twocomponent flavin-dependant monooxygenase family, such as styrene monooxygenase from Pseudomonas Putida S12 (37), the HPAH-C 2 monooxygenase from A. baumannii (22), and bacterial luciferase (38). The recently solved x-ray structure of the HPAH-C 2 monooxygenase from A. baumannii in complex with FMN red has revealed the presence of a cavity adjacent to the isoalloxazine ring of the flavin molecule that is appropriate for harboring the oxygen atoms of the C(4a)-FMN-OOH intermediate (39).…”

“…2B) that has an additional band extending to 600 nm. This spectrum is similar to that of a mixture of ϳ10 M FMN ox and ϳ10 M DHK ox -OH; the latter spectrum was presented in our previous work (24). Oxidation of hydroquinones and their derivatives such as DHK red by O 2 appears to proceed through an autocatalytic mechanism with phases that are only partially dependent on the concentration of O 2 (28,32,33).…”

“…In this work, we have carried out a detailed kinetic study of the reaction of the two-component FMN-dependent monooxygenase, ActVA-ActVB from S. coelicolor, by stopped-flow spectrophotometric methods to test our earlier propositions on the reaction mechanism of ActVA-ActVB system (17,24) and to gain insights into its mechanism of regulation. Recent characterization of several members of the two-component flavin-dependent monooxygenase family has revealed similarities to but also some specific differences from the well studied single component flavoprotein monooxygenase family, such as p-hydroxybenzoate hydroxylase.…”

“…Intermediate-It has been previously shown that ActVA is able to stabilize a C(4a)-FMN-OOH species within its active site, but the kinetics of its formation and decay were not determined (17,24). Here, we have investigated the kinetics of the formation and decay of the C(4a)- (22) and p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa (25,26).…”