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Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of [FADH.sub.2] provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when [FADH.sub.2] is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of [FADH.sub.2], resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the [FADH.sub.2]-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotech. nological applications and is highly relevant from a structural and evolutionary point of view. doi: 10.1128/JB.00723-10

How instructive cues present on the cell surface have their precise effects on the actincytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer (1). Semaphorins/collapsins were characterized in part on the basis of their ability to drastically alter actin cytoskeletal dynamics in neuronal processes (2), but despite considerable progress in the identification of semaphorin receptors and their signalling pathways (3), the molecules linking them to the precise control of cytoskeletal elements remain unknown. Recently, highly unusual proteins of the Mical family of enzymes have been found to associate with the cytoplasmic portion of plexins, which are large cell-surface semaphorin receptors, and to mediate axon guidance, synaptogenesis, dendritic pruning and other cell morphological changes (4-7). Mical enzymes perform reduction-oxidation (redox) enzymatic reactions (4,5,8-10) and also contain domains found in proteins that regulate cell morphology (4,11). However, nothing is known of the role of Mical or its redox activity in mediating morphological changes. Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics. We found that Mical is both necessary and sufficient for semaphorin-plexin-mediated F-actin reorganization in vivo. Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments. We also found that Mical utilizes its redox activity to alter F-actin dynamics in vivo and in vitro, indicating a previously unknown role for specific redox signalling events in actin cytoskeletal regulation. Mical therefore is a novel F-actin-disassembly factor that provides a molecular conduit through which actin reorganization--a hallmark of cell morphological changes including axon navigation--can be precisely achieved spatiotemporally in response to semaphorins., Multidomain cytosolic proteins of the Mical family (Fig. 1a) mediate semaphorin--plexin repulsive axon guidance and cell morphological changes4,5, but their role in controlling these events is unknown. While characterizing new [...]

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of [His.sub.10]-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(H) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate ([K.sub.i] = 14 [micro]M) was competitive with hydroquinone. Online size-exclusion chromatographymass spectrometry revealed that HQDO is an [alpha]2[beta]2 heterotetramer of 112.4 kDa, which is composed of an [alpha]-subunit of 17.8 kDa and a [beta]-subunit of 38.3 kDa. Each [beta]-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.

The catabolism of 4-hydroxyacetophenone in Pseudomonasfluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to [beta]-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Ycil family (hapH), and a [2Fe-2S] ferredoxin (hapl) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.

The rho-hydroxybenzoate hydroxylase (PHBH)-mediated conversion of tetrafluoro-4-hydroxybenzoate (F4-POHB) involves a number of consecutive oxygenation and dehalogenation steps. These include hydroxylation at the substrate C2 position. The enzymatic conversion of F3-DOHB to 5,6-F2-TOHB implies that F3-DOHB binding leads to the stabilization of the flavin C(4a)-hydroperoxide. It also indicates that the peroxide bond of the oxygenated flavin intermediate becomes oriented in such a way as to allow C2 hydroxylation.

The growth of Penicillium simplicissimum on anisyl alcohol, veratryl alcohol or 4-(methoxymethyl)phenol causes the formation of the covalent flavoprotein, vannillyl-alcohol oxidase. The induction of the enzyme is highest during the growth phase, and is suppressed by the addition of isoeugenol to cultures containing veratryl alcohol. However, the formation of the enzyme is nonessential, as the degradation pathways of veratryl and anisyl alcohols have no requirement for vannillyl-alcohol oxidase. The enzyme is involved in the metabolism of 4-(methoxymethyl)phenol and p-cresol methylether.