Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

Abstract

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 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.The incorporation of one atom of oxygen during hydroxylation, epoxidation, sulfoxidation, or Baeyer-Villiger oxidation is a common initial step of the aerobic degradation of aromatic compounds by microorganisms. In bacteria, these reactions are most frequently catalyzed by inducible flavoprotein monooxygenases (EC 1.14.13 [57]). The majority of these enzymes (socalled single-component flavoprotein monooxygenases) utilize electrons from NAD(P)H, which are transferred to a noncovalently bound flavin adenine dinucleotide (FAD) in order to activate molecular oxygen as a flavin (hydro)peroxide. Depending on the protonation of this intermediate and the type of substrate, an oxygen atom is then incorporated by nucleophilic or electrophilic attack. More recently, different twocomponent flavoprotein monooxygenases have been characterized (57). These systems cover an NAD(P)H-dependent flavin reductase in order to generate reduced flavin and an oxygenase that utilizes this cofactor for the activation of oxygen.The exquisite regio-and stereoselectivities of oxygen insertion by flavoprotein monooxygenases favor these enzymes for biocatalytic applications (23,24,33). This is especially true because chemical synthesis approaches by hetero-or homogenic catalysis often do not yield a sufficiently high enantiomeric excess for the production of pharmaceuticals and their chiral building blocks. The use of oxygen as an inexpensive nontoxic oxidant and mild reaction conditions are additional advantages with the potential for increasing the environmental sustainability of oxygenase-catalyzed bio...

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

“…Tyr and Ser, respectively. However, it is noteworthy that self-sufficient one-component SMOs have Ile and Ala at the corresponding positions, respectively, which includes the StyA2B from Rhodococcus opacus 1CP (Tischler et al, 2009) and two putative SMOs from Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 (Fig. 5).…”

“…Therefore, we assumed that the putative active cavity of SMO should be strictly shaped by surrounding residues and the mutations could hardly change the orientation of the substrate. The reversal of enantioselectivity for the mutant Y73V during the epoxidation of 1-phenylcyclohexene was unexpected because all other experimentally assigned SMOs display the same enantioselectivity (Bernasconi et al, 2000;Di Gennaro et al, 1999;Lin et al, 2010;Panke et al, 1998;Park et al, 2005;Tischler et al, 2009;van Hellemond et al, 2005), and the overall structure of the active cavity of SMOs should not be flexible enough to generate complementary enantiomers. Moreover, the mutants from directed evolution were also reported to produce (S)-enantiomers (Gursky et al, 2010).…”

StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System

“…Moreover, the relaxed substrate specificity of SMOs allows an enantioselective conversion of substituted styrene derivatives and structurally related compounds, like indene and dihydronaphthalene, as well as phenylalkylsulfides ( Fig. 1) (17,40,45), thus increasing their biocatalytic potential.…”

Isolation and Characterization of a 4-Nitrotoluene-Oxidizing Enzyme from Activated Sludge by a Metagenomic Approach

“…The deduced product of icpB (172 amino acids) was 38% identical to a flavin reductase related to styrene's oxygenation from Rhodococcus opacus 1CP (48). The IcpB protein also showed homology (38% identity) to a NADH:Flavin mononucleotide oxidoreductase involved in the oxygenation of nitrilotriacetate Chelatobacter heintzii ATCC29600 (51).…”

“…Furthermore, the flavin reductase-like protein (icpB) was found downstream from the icpA gene. The deduced product of icpB (174 amino acids) showed 41% identity to the flavin reductase protein related to styrene oxygenation from Rhodococcus opacus 1CP (48). Sequencing of the fosmid pM123 revealed an insert of 35 kb and the presence of 31 ORFs.…”