Janosch Gröning

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

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 FADH2 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 FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-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.

One-component styrene monooxygenases: an evolutionary view on a rare class of flavoproteins.

Styrene monooxygenases (SMOs) are catalysts for the enantioselective epoxidation of terminal alkenes. Most representatives comprise a reductase and a monooxygenase which are encoded by separate genes (styA, styB). Only six presumed self-sufficient one-component SMOs (styA2B) have previously been submitted to databases, and one has so far been characterized. StyA2B can be supported by another epoxidase (StyA1) encoded by styA1, a gene in direct neighborhood of styA2B. The present report describes the identification of a further styA1/styA2B-like SMO, which was detected in Rhodococcus opacus MR11. Based on the initially available sequences of styA2B-type SMOs, primers directed at conserved sequences were designed and a 7,012-bp genomic fragment from strain MR11 was obtained after PCRs and subsequent genome walking. Six open reading frames (ORFs) were detected and compared to genomic fragments of strains comprising either two- or one-component SMOs. Among the proteins encoded by the ORFs, the monooxygenase StyA1/StyA2B showed the highest divergence on amino acid level when comparing proteins from different sources. That finding, a rare distribution of styA2B genes among bacteria, and the general observation of evolution from simple to complex systems indicate that one-component SMOs evolved from two-component ancestors. Analysis of gene products from styA/styB- and styA1/styA2B-like SMOs revealed that a fusion of styA/styB to styA2B might have happened at least twice among microorganisms. This points to a convergent evolution of one-component SMOs.

Two unusual chlorocatechol catabolic gene clusters in Sphingomonas sp. TFD44

The genes responsible for the degradation of 2,4-dichlorophenoxyacetate (2,4-D) by α-Proteobacteria have previously been difficult to detect by using gene probes or polymerase chain reaction (PCR) primers. PCR products of the chlorocatechol 1,2-dioxygenase gene, tfdC, now allowed cloning of two chlorocatechol gene clusters from the Sphingomonas sp. strain TFD44. Sequence characterization showed that the first cluster, tfdD,RFCE, comprises all the genes necessary for the conversion of 3,5-dichlorocatechol to 3-oxoadipate, including a presumed regulatory gene, tfdR, of the LysR-type family. The second gene cluster, tfdC2E2F2, is incomplete and appears to lack a chloromuconate cycloisomerase gene and a regulatory gene. Purification and N-terminal sequencing of selected enzymes suggests that at least representatives of both gene clusters (TfdD of cluster 1 and TfdC2 of cluster 2) are induced during the growth of strain TFD44 with 2,4-D. A mutant constructed to contain an insertion in the chloromuconate cycloisomerase gene tfdD still was able to grow with 2,4-D, but more slowly and with a longer lag phase. This, and the detection of additional activity peaks during protein purification suggest that strain TFD44 harbors at least another chloromuconate cycloisomerase gene. The sequence of the tfdCE region was almost identical to that of a partially characterized chlorocatechol catabolic gene cluster of Sphingomonas herbicidovorans MH, whereas the sequence of the tfdC2E2F2 cluster was different. The similarity of the predicted proteins of the tfdD,RFCE and tfdC2E2F2 clusters to known sequences of other Proteobacteria in the database ranged from 42 to 61% identical positions for the first cluster and from 45.5 to 58% identical positions for the second cluster. Between both clusters, the similarities of their predicted proteins ranged from 44.5 to 64% identical positions. Thus, both clusters (together with those of S. herbicidovorans MH) represent deep-branching lines in the respective dendrograms, and the sequence information will help future primer design for the detection of corresponding genes in the environment.

Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

ABSTRACT Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process.

Gene redundancy of two-component (chloro)phenol hydroxylases in Rhodococcus opacus 1CP

Among other factors, a distinct gene redundancy is discussed to facilitate high metabolic versatility of rhodococci. Rhodococcus opacus 1CP is a typical member in that respect and degrades a multitude of (chlorinated) aromatic compounds. In contrast to the central pathways of aromatic degradation in strain 1CP, little is known about the degree of gene redundancy and to what extent this is reflected on protein level within the steps of peripheral degradation. By means of degenerated primers deduced from tryptic peptides of a purified phenol hydroxylase component and using the amplified fragment as a labelled probe against genomic 1CP-DNA, three gene sets encoding three different two-component phenol hydroxylases pheA1/pheA2(1-3) could be identified. One of them was found to be located on the megaplasmid p1CP, which confirms the role of these elements for metabolic versatility. Protein chromatography of phenol- and 4-chlorophenol-grown 1CP-biomass gave first evidences on a functional expression of these oxygenases, which could be initially characterised in respect of their substrate specificity.

Recombinant expression of a unique chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP and identification of catalytically relevant residues by mutational analysis

Chloromuconolactone dehalogenase ClcF plays a unique role in 3-chlorocatechol degradation by Rhodococcus opacus 1CP by compensating the inability of its chloromuconate cycloisomerase ClcB2 to dechlorinate the chemically stable cycloisomerization product (4R,5S)-5-chloromuconolactone (5CML). High sequence similarities showed relatedness of ClcF to muconolactone isomerases (MLIs, EC 5.3.3.4) of the 3-oxoadipate pathway. Although both enzyme types share the ability to dechlorinate 5CML, comparison of kcat/Km indicated a significant extent of specialization of ClcF for dechlorination. This assumption was substantiated by an almost complete inability of ClcF to convert (4S)-muconolactone and the exclusive formation of cis-dienelactone from 5CML. Mutational analysis of ClcF by means of variants E27D, E27Q, Y50A, N52A, and A89S indicated relevance of some highly conserved residues for substrate binding and catalysis. Based on the putative isomerization mechanism of MLI, evidence was provided for a role of E27 in initial proton abstraction as well as of Y50 and N52 in substrate binding. In case of N52 substrate binding is likely to occur to the carboxylic group of 5CML as indicated by a significant change of product specificity. Expression in Escherichia coli BL21-CP(DE)-RIL followed by a three-step purification procedure with heat treatment is a convenient strategy to obtain recombinant ClcF and variants thereof.

Crystal structure and catalytic mechanism of chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP

The actinobacterium Rhodococcus opacus 1CP possesses a so far unique variant of the modified 3‐oxoadipate pathway for 3‐chlorocatechol degradation. One important feature is the novel dehalogenase ClcF, which converts (4R,5S)‐5‐chloromuconolactone to E‐dienelactone. ClcF is related to muconolactone isomerase (MLI, EC 5.3.3.4). The enzyme has a ferredoxin‐type fold and forms a homodecamer of 52‐symmetry, typical for the MLI family. The active site is formed by residues from two monomers. The complex structure of an E27A variant with bound substrate in conjunction with mutational studies indicate that E27 acts as the proton acceptor in a univalent single‐base syn‐dehydrohalogenation mechanism. Despite the evolutionary specialization of ClcF, the conserved active‐site structures suggest that the proposed mechanism is representative for the MLI family. Furthermore, ClcF represents a novel type of dehalogenase based on an isomerase scaffold.

Crystallization and preliminary characterization of chloromuconolactone dehalogenase from Rhodococcus opacus 1CP.

Chloroaromatic compounds are often very persistent environmental pollutants. Nevertheless, numerous bacteria are able to metabolize these compounds and to utilize them as sole energy and carbon sources. Rhodococcus opacus 1CP is able to degrade several chloroaromatic compounds, some of them via a variation of the 3-chlorocatechol branch of the modified ortho-cleavage pathway. This branch in R. opacus differs from that in Proteobacteria in the inability of the chloromuconate cycloisomerase to dehalogenate. Instead, a unique enzyme designated as chloromuconolactone dehalogenase (ClcF) is recruited. ClcF dehalogenates 5-chloromuconolactone to cis-dienelactone and shows a high similarity to muconolactone isomerases (EC 5.3.3.4). However, unlike the latter enzymes, it is unable to catalyse the isomerization of muconolactone to 3-oxoadipate enollactone. In order to characterize the catalytic mechanism of this unusual dehalogenase, the enzyme was crystallized and subjected to X-ray structural analysis. Data sets to up to 1.65 Å resolution were collected from two different crystal forms using synchrotron radiation. Crystal form I (space group P2(1)) contained 40 subunits in the asymmetric unit, whereas ten subunits were present in crystal form II (space group P2(1)2(1)2(1)). The self-rotation function revealed the orientations of the molecular symmetry axes of the homodecamer of 52 symmetry.

Optimization of a genome-walking method to suit GC-rich template DNA from biotechnological relevant Actinobacteria.

SiteFinding‐PCR has been recently reported to be a useful technique in order to identify unknown DNA fragments located adjacent to available sequences. However, this method has so far only been applied to few DNA sources including plants, samples from bioleaching communities, and a Pseudomonas strain. In order to complete the sequence information of two gene clusters in Gram‐positive rhodococci the original protocol was applied yielding amplicons of insufficient size. The binding site of the previously published SiteFinder‐2 oligo proved to be unsuitable for Rhodococcus and other members of the Actinobacteria since the binding motif occurred too frequently. Available genome sequences of different Actinobacteria were analysed and the binding site of the SiteFinder oligo modified. Moreover, PCR conditions were adapted to the high GC content of the template DNA allowing the successful adaptation of this method to two members of the Actinobacteria. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Draft genome sequence of Rhodococcus erythropolis B7g, a biosurfactant producing actinobacterium

Biosurfactants are amphipathic molecules with relevance in biotechnology due to their structural diversity, low toxicity and biodegradability. The genus Rhodococcus has extensively been studied because of its capacity to produce trehalose-containing surfactants as well as trehalose lipids as potential pathogenic factor. Here we present the draft genome sequence of Rhodococcus erythropolis B7g isolated with toluene from fuel-contaminated soil. The genome comprises 7,175,690 bp in 121 contigs, a G + C content of 62,4% and 7,153 coding DNA sequences (CDSs), and it contains genes for trehalose biosynthesis and surfactant production. Additionally, genes for the production of trehalose-tetraester biosurfactant were identified, whose function was experimentally verified making the strain B7g a potential candidate for use in bioremediation applications or in biosurfactant exploration.