Monooxygenase alkene

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous B-276 (also known as Nocardia corallina) is a multicomponent oxygenase that catalyzes the stereoselective insertion of an oxygen atom from O2 into aliphatic alkenes, yielding the corresponding chiral epoxides (Table 1) [151], This reaction is the first step in the pathway of the alkene metabolism. AMO utilizes substrates ranging from C3- 

EPR techniques have proven pivotal in establishing the identity of the pros-thetie groups and the determination of the midpoint potential of the reduetase eomponent of AMO [159], From these studies the following mechanism for the elee- 

However, in contrast to MMO, AMO is not inhibited by ethyne, and its reactivity is only mildly affected by the presence of propyne [16]. Since both enzymes also display different stereochemistries despite having homologous active sites, it has been speculated that MMO and AMO employ alternative catalytic mechanisms [16]. While the observation of AMO activation by hydrogen peroxide [160] suggests the formation of a possible diiron-peroxo intermediate in the reaction cycle, the enzyme s inability to oxidize methane indicates that no Q-like diferryl-oxo species may be formed [98]. Hence, small differences in the coordination environment of the diiron centers in AMO and MMO lead to significant variations in substrate specificity and reactivity. 

NATASA MITIC, GERHARD SCHENK AND GRAEME R. HANSON 

5 Miscellaneous Iron-Sulfur Flavoproteins. 5.5.1 Alkene monooxygenase. Nocardia corallina B-276 possesses a multi-component enzyme, alkene monooxygenase, that catalyses the stereoselective epoxygenation of alkenes to give predominantly the R enantiomer. 

Gallagher and co-workers have characterized the reductase component by EPR and fluorescence spectroscopy. They showed that it contained one FAD and a [IFe-lS] cluster. The FAD could be reduced in a two-step reaction to the fully reduced flavin. The optical spectrum of the semiquinone species was typical of a neutral flavin radical. The [2Fe-2S] + cluster could also be reduced by one electron equivalent to [2Fe-2S] +. Both paramagnetic species could be detected by EPR. It could also be shown by a combination of mid-point potential measurements and electron-transfer kinetics that this component could supply the energy required for the epoxygenation reaction. 

Thus the authors were able to suggest a mechanism for electron transfer within the alkene monooxygenase complex, in which the FAD moiety is reduced to FADH by bound NADH. The electrons are then transported one at a time to the [2Fe-2S] cluster, which in turn reduces the di-iron centre of the epoxygenase. The reduction of the di-iron centre is required for the binding of molecular oxygen and subsequent substrate oxidation. 

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Hartmans S, FJ Weber, BPM Somhorst, JAM de Bont (1991) Alkene monooxygenase from Mycobacterium E3 a multicomponent enzyme. J Gen Microbiol 137 2555-2560. 

Zhou N-Y, A Jenkins, CKN Chan, KW Chion, DJ Leak (1999) The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monooxygenation of benzene, toluene, and phenol. Appl Environ Microbiol 65 1589-1595. 

Ensign SA (1996) Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2. Appl Environ Microbiol 62 61-66. 

Small FJ, SA Ensign (1997) Alkene monooxygenase from Xanthobacter strain Py2. Purification and characterization of a four-component system central to the bacterial metabolism of aliphatic alkenes. J Biol Chem 272 24913-24920. 

The aerobic degradation of chloroethene (vinyl chloride) by Mycobacterium aurum strain LI proceeded by initial formation of an epoxide mediated by an alkene monooxygenase (Hartmans and de Bout 1992). This reaction has also been demonstrated to occur with Methylosinus trichosporium, even though subsequent reactions were purely chemical (Castro et al. 1992b). 

Prichanont, S., Leak, D.J. and Stuckey, D.C. (1998) Alkene monooxygenase-catalyzed whole cell epoxidation in a two-liquid phase system. Enzyme and Microbial Technology, 22 (6), 471 479. 

Ensign, S. A., Hyman, M. R. Arp, D. J. (1992). Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain. Applied and Environmental Microbiology, 58, 3038-46. 

Chiral epoxide Alkene monooxygenase Nocardia corallina... 

In Xanthobacter sp. strain Py2 both the alkene monooxygenase and the epoxidase are induced by C2, C3, and C4 alkenes, and also by chlorinated alkenes including vinyl chloride, cis- and trans-dichlo-roethene and 1,3-dichloropropene (Ensign 1996). 

Indeed, vinyl chloride- and etiiene-grown cells of strain LI have been shown to oxidize propene to 1,2-epoxypropane [30], indicating that alkene monooxygenase was present in cells grown on both the substrates. 

NADH- and os gen-dependent alkene monooxygenase was also detected in dialyzed crude extracts of ethene- and vinyl chloride>grown cells [26]. No monooxygenase activity was detected in cells grown on acetate or succinate. 

The presence of alkene monooxygenase activity in extracts of vinyl chloride-grown M. aurum LI, combined witli the observation that vinyl chloride degradation can be competitively inhibited by the addition of ethene or propene, which are both oxidized to the corresponding epoxides, is strong evidence that the initial step in vinyl chloride metabolism is indeed catalyzed by alkene monooxygenase (Figure 3) [25]. 

Figure 3. The initial step in vinyl chloride metabolism of M, aurum LI, catalyzed by alkene monooxygenase.

In studying the inhibitory effects of the less reactive l,2-epo]ypropane, it was previously demonstrated that the inhibitory effect on the monooiygenases examined was much stronger than on other physiological functions of the cell [31]. Althouf not examined for strain LI, this is probably also the case with alkene monooxygenase inactivation by chlorooxirane. 

Gallagher SC, Cammack R, Dalton H. 1997. Alkene monooxygenase from Nocardia corallina B-276 is a member of the class of dinuclear iron proteins capable of stereospecific epoxygenation reactions. Eur JBiochem 247 635-641. 

Zhou NY, Jenkins A, Chion C, Leak DJ. 1998. The alkene monooxygenase from Aon-thobacter Vy2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase. FEBS Lett 430 181-185. 

Miura A, Dalton H. 1995. Purification and characterization of the alkene monooxygenase from Nocardia corallina B-276. Biosci Biotechnol Biochem 59 853-859. 

Saeki H, Akira M, Furuhashi K, AverhoffB, Gottschalk G. 1999. Degradation of tri-chloroethene by a hnear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus Nocardia corallina) B-276. Microbiology-UK 145 1721-1730. 

Smith TJ, Lloyd JS, Gallagher SG, Fosdike WLJ, Murrell JG, Dalton H. 1999. Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276. Eur J Biochem 260 446 52. 

Saeki H, Furuhashi K. 1994. Gloning and characterisation of a Nocardia corallina B-276 gene cluster encoding alkene monooxygenase. J Ferment Bioeng 78 399 06. 

Fosdike WLJ, Smith TJ, Dalton H. 2005. Adventitious reactions of alkene monooxygenase reveal common reaction pathways and component interactions among bacterial hydrocarbon oxygenases. F 557272 2661-2669. 

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