The ‘iron bacteria’ are a collection of morphologically and phylogenetically heterogeneous prokaryotes. They include some of the first micro-organisms to be observed and described, and continue to be the subject of a considerable body of fundamental and applied microbiological research. While species of iron-oxidizing bacteria can be found in many different phyla, most are affiliated with the Proteobacteria. The latter can be subdivided into four main physiological groups: (i) acidophilic, aerobic iron oxidizers; (ii) neutrophilic, aerobic iron oxidizers; (iii) neutrophilic, anaerobic (nitrate-dependent) iron oxidizers; and (iv) anaerobic photosynthetic iron oxidizers. Some species (mostly acidophiles) can reduce ferric iron as well as oxidize ferrous iron, depending on prevailing environmental conditions. This review describes what is currently known about the phylogenetic and physiological diversity of the iron-oxidizing proteobacteria, their significance in the environment (on the global and micro scales), and their increasing importance in biotechnology.

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...

Recently, combined carbon and hydrogen isotope fractionation investigations have emerged as a powerful tool for the characterization of reaction mechanisms relevant for the removal of organic pollutants. Here, we applied this approach in order to differentiate benzene biodegradation pathways under oxic and anoxic conditions in laboratory experiments. Carbon and hydrogen isotope fractionation of benzene was studied with four different aerobic strains using a monooxygenase or a dioxygenase for the initial benzene attack, a facultative anaerobic chlorate-reducing strain as well as a sulfate-reducing mixed culture. Carbon and hydrogen enrichment factors (epsilon(C), epsilon(H)) varied for the specific pathways and degradation conditions, respectively, so that from the individual enrichment factors only limited information could be obtained for the identification of benzene biodegradation pathways. However, using the slope derived from hydrogen vs carbon isotope discriminations or the ratio of hydrogen to carbon enrichment factors (lambda = deltaH/ deltaC approximately epsilon(H)/epsilon(C)), benzene degradation mechanisms could be distinguished. Although experimentally determined lambda values partially overlapped, ranges could be determined for different benzene biodegradation pathways. Specific lambda values were < 2 for dihydroxylation, between 7 and 9 for monohydroxylation, and > 17 for anaerobic degradation. Moreover, variations in lambda values suggest that more than one reaction mechanism exists for monohydroxylation as well as for anaerobic benzene degradation under nitrate-reducing, sulfate-reducing, or methanogenic conditions. Our results show that the combined carbon and hydrogen isotope fractionation approach has potential to elucidate biodegradation pathways of pollutants in field and laboratory microcosm studies.

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 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 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 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 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 biotechnological applications and is highly relevant from a structural and evolutionary point of view.The environmentally harmful hydrocarbon styrene is readily biodegradable by various classes of microorganisms covering Gram-negative and Gram-positive bacteria as well as fungi (e.g., ascomycetes). Two major pathways for styrene mineralization have been described (reviewed in references 23, 26, and 33); of these, the most common one is initiated by a monooxygenase-catalyzed epoxidation of the vinyl side chain. Due to their biotechnological potential, the styrene monooxygenases (SMOs) involved in this reaction have received considerable attention. Most SMOs have been described for pseudomonads and were investigated for their biochemical properties (5,13,27,31,46) and their biotechnological applicability in cell-free (16, 17) or whole-cell systems (3,12,28,29,30,32,37). All SMOs investigated thus far convert styrene in a highly enantioselective manner to (S)-styrene oxide, which is a useful precursor for several chiral synthons and pharmaceuticals (2,6,14,26,34). 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.Typical SMOs of pseudomonads consist of two enzymatically active protein components encoded by genes that are usually clustered adjacent to each other (styA and styB) (Fig. 2a) (26,43,46). The flavin reductase subunit (StyB) reduces flavin adenine dinucleotide (FAD) at the expense of NADH.The monooxygenase subunit (StyA) then utilizes the reduced flavin (FADH 2 ) to activate molecular oxygen for styrene attack (Fig. 2b)....

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.