Methyl Group Hydroxylation versus Addition to Fumarate. INTRODUCTIONBiological degradation of aromatic hydrocarbons is challenged by their extraordinary chemical stability arising from the exclusive. C—C and C—H bonds and the resonance stabilization of the aromatic ring (1). Aerobic bacteria employ a variety of oxygen- dependent enzymes, i. One such enzyme is the membrane- bound methyl- hydroxylating toluene/xylene monooxygenase (Xyl. M) encoded on the TOL plasmid. Pseudomonas putida mt- 2 (3). Xyl. M is a non- heme di- iron monooxygenase, which forms (methyl)benzyl alcohol from (alkyl)toluene. Likewise, a bifunctional. Cym. Aa, hydroxylase subunit; Cym. Ab, reductase subunit) of P. Thus, hydroxylation of benzylic methyl groups represents one important mode for the activation of aromatic hydrocarbons. However, due to the strict O2 dependence of these enzymes, this reaction is not feasible for bacterial oxidation of such substrates under anoxic conditions. The addition of toluene to fumarate, yielding benzylsuccinate by an enzymatic radical reaction, was originally discovered. Insights into interfacial activation from an open structure of Candida rugosa lipase. How do serine proteases really work Chiral. Conversion of crude oil to methane by a microbial consortium enriched from oil reservoir production waters. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2014.00197 Microbiology Original Research Article Conversion of crude oil to methane by a microbial consortium enriched from oil reservoir production waters Berdugo-Clavijo Carolina Gieg. Microbial Hydrocarbon Degradation: Efforts to Understand Biodegradation in Petroleum Reservoirs http://dx.doi.org/10.5772/55920. A closely evolutionarily related enzyme in the AP superfamily is the arylsulfatase from Pseudomonas aeruginosa (PAS) (Figure 1C). This enzyme only shares about 27% sequence similarity to RlPMH but has high structural similarity, in that 64% of the residues. Thauera aromatica K1. T (1. 1) and Azoarcus sp. This type of reaction proved to be archetypical among diverse facultatively and obligately anaerobic bacteria for the initial. In the case of the latter, addition to fumarate is only known for energy- limited sulfate- reducing bacteria, while denitrifying. Aromatoleum aromaticum” strains Eb. N1 and EB1 activate ethylbenzene via hydroxylation to (S)- 1- phenylethanol (2. Taken together, benzylic methyl groups in aromatic hydrocarbons are activated only via addition to fumarate in all described. O2- independent hydroxylation has not been demonstrated to date. In contrast, both reaction types have been reported for the. P. In the case of (alkyl)toluene- derived (alkyl)benzylsuccinate(s), a modified . In contrast, conversion of ethylbenzene- derived (S)- 1- phenylethanol to benzoyl- Co. A in “A. Benzoyl- Co. A is further metabolized to acetyl- Co. A via a central degradation pathway involving reductive dearomatization. The bacterial strains investigated in this study, “A. In contrast to strain p. Cy. N2, however, strain p. Cy. N1 also anaerobically grows with 4- ethyltoluene and toluene (Table 1). Both strains were cultivated under nitrate- reducing conditions (7 m. M nitrate) and an anoxic atmosphere of N2- CO2 (9. M) and bicarbonate- buffered defined mineral medium (3. Isopropylbenzoate (2 m. M), succinate (5 m. M), or 2,3- 2. H2- fumarate (1. M) was added from sterile aqueous stock solutions. Growth was determined by measuring the optical density at. OD6. 60) in a spectrophotometer (UV- 1. Shimadzu, Kyoto, Japan). Prior to cultivation for DNA sequencing or proteomic or metabolite. A. Six parallel cultures (4. OD6. 60s of 0. 2. A. In addition, such cultivations were also carried out with 4- ethyltoluene, toluene. H2- fumarate in the case of “A. The cultures were inactivated by incubation at 8. To avoid interferences from coeluting compounds, the extract obtained upon growth. A. GC- MS measurements were performed using a Trace GC- MS (Thermoelectron, Dreieich. Germany). The GC was equipped with a temperature- programmable injection system and a BPX5- fused silica capillary column (length. Helium was used as the carrier gas at a constant flow rate. The GC oven temperature was programmed from 5. The MS was operated in electron ionization mode at an ion source temperature of 2. Full- scan mass spectra were recorded. Da at a rate of 2. Identification of metabolites was based on comparison of GC retention times and mass spectra with those of authentic standards. For stereochemical assignment, arylsuccinates were derivatized with (R)- 1- phenylethanamine and analyzed as recently described by Jarling et al. Isoelectric focusing was performed using 2. H gradient (IPG) strips with a nonlinear (NL) p. H range. from 3 to 1. GE Healthcare, Munich, Germany) in an IPGphor system (GE Healthcare). Separation according to molecular mass. Ettan Dalt II system (GE Healthcare). Cy. Dye DIGE Fluor dyes (2. Protein extracts from p- cymene- grown cells served as test states and were labeled with Cy. Protein extracts of succinate- grown cells served as the. Cy. 5. The internal standard comprised equal amounts of extracts of p- cymene- and succinate- grown cells, which were labeled with Cy. Each individual gel was loaded with equal amounts of the. Four gels were prepared per strain, with each gel comprising the. Digital gel images were directly acquired after electrophoresis. Typhoon 9. 40. 0 scanner (GE Healthcare) and analyzed with De. Cyder software (version 7. GE Healthcare). Abundance changes. Protein spots specifically formed in p- cymene- adapted cells were excised with an EXQuest spot cutter (Bio- Rad, Munich, Germany), washed, and digested as described. Tryptic peptides were spotted onto Anchorchip steel targets (Bruker Daltonik Gmb. H, Bremen, Germany) and analyzed with an. Ultrafle. Xtreme matrix- assisted laser desorption ionization–two- stage time of flight (MALDI- TOF/TOF) mass spectrometer (Bruker. Daltonik Gmb. H) (4. Each sample lane was cut in four slices, and each slice was cut into . Generated peptides were separated with an Ulti. Mate 3. 00. 0 RSLCnano system (Thermo Fisher Scientific, Germering, Germany). C1. 8; pore size, 1. Continuous analysis of the eluent was performed with an online- coupled ion trap mass spectrometer (ama. Zon ETD; Bruker Daltonik. Gmb. H) operated as described previously (4. Acquired mass spectra were searched against the translated. A. Identified peptides of all samples per strain and growth condition were compiled. Open reading frame (ORF) finding and automatic functional assignment involved the HTGA (High- Throughput Genome Annotation). RAST (Rapid Annotation using Subsystem Technology) (4. ARTEMIS (4. 8) was used to manually refine the start position of predicted ORFs. Refinement of functional predictions of identified proteins. BLAST function of the Uni. Prot knowledgebase (Uni. Prot. KB . First, selected protein sequences were aligned with Clustal W (5. Second, based on the Clustal W- generated alignments, Meg. Align constructs rooted trees using 1,0. This is indicated by NA (not. The degradation. pathways reconstructed on the basis of these findings are depicted in Fig. Compounds shown in black represent proven growth substrates. Protein and gene names are shown in black (identified). Identified proteins are detailed in Table 2 as well as in Table S1 and Fig. S2 in the supplemental material (except for 1. Numbered structures correspond. Co. A; 9, (E)- (4- isopropylphenyl)itaconyl- Co. A; 1. 0, 2- . Identified metabolites are marked with an asterisk (*) and are detailed in Fig. S1 in the supplemental material); 4- isopropylbenzoate was also identified in the case of “Thauera” sp. In addition, a metabolite was detected. Krieger et al. These findings indicate that “A. Furthermore, all necessary genes for this degradation pathway (e. Bss. A displaying 1. Eb. N1) were detected in the genome of “A. Rabus, unpublished data). During growth with. Thauera” sp. Subsequently, the metabolite was unequivocally identified as dimethyl (4- isopropylbenzyl)succinate by comparison with a. Derivatization of the free acid with (R)- 1- phenylethanamine enabled separation of the racemate. As known for toluene- derived benzylsuccinate (5. R- configuration. During anaerobic growth of “Thauera” sp. S1 in the supplemental material). This provided direct evidence for involvement of. Furthermore, a metabolite was detected, the mass spectrum of which agrees with the structure of (4- isopropylphenyl)itaconate. S1 in the supplemental material). Another metabolite with a molecular ion at m/z 1. Fig. Overall, these results provide evidence that p- cymene is anaerobically degraded in “Thauera” sp. Likewise, 4- ethylbenzoate (Fig. C) was formed with 4- ethyltoluene as the substrate. Unexpectedly, however, no evidence was obtained for the formation of the. Instead, the respective benzyl alcohols and benzaldehydes. Fig. 2. A and B and 3. A and B). These findings provide direct metabolic evidence for the O2- independent dehydrogenation/oxidation of a benzylic methyl group as the initial activation reaction during bacterial utilization. S2 in the supplemental material. The functional predictions of these proteins are compiled in Table S1 in the supplemental. The proteogenomic findings are summarized in Fig. Colocalization of genes and homology of protein sequences, as well as key features (see Fig. S3 in the supplemental material). Cmd. ABC from “A. The large subunit Cmd. A possesses a twin- arginine motif (R7. R8) as part of an N- terminal signal peptide, suggesting translocation of the folded protein via the Tat- secretion pathway (5. Moreover, Cmd. A contains the conserved Asp. Lys. 43. 4 residues, which were previously implicated for ethylbenzene dehydrogenase to be involved in coordination of the Mo- bis- molybdopterin guanine dinucleotide (Mo- bis. MGD) cofactor in the active site and the conserved binding site for the FS0- . The Cmd. B subunit harbors a series of conserved cysteine residues for binding of a total of four Fe- S clusters, and the. Met. 10. 4 and Lys. Cmd. C subunit may function as the two axial ligands of the heme- Fe (5. Based on these findings, the presumptive p- cymene dehydrogenase (Cmd. ABC) is assumed to catalyze the O2- independent hydroxylation of p- cymene to 4- isopropylbenzyl alcohol. Cmd. A and Ibs. A are marked in bold and highlighted in gray. Sequence identities (percentages) are based on. Clustal W. Open circles indicate > 7. The bar below each tree corresponds. The red star marks carbon atoms targeted during the initial activation. C6 to C1. 6 (7). Abbreviations of enzyme catalytic subunits in panel A are as follows: S2. A, steroid C- 2. 5 dehydrogenase; S2. A2- 7, steroid. C- 2. Hd. N1. F. Abbreviations of enzymes (catalytic subunits) in panel B are as follows: Bss. A and Tut. D, benzylsuccinate synthase. Hbs. A, (hydroxybenzyl)succinate synthase; Nms. A, (2- naphthylmethyl)succinate synthase; Ass.
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