Glycyl Radical Research Articles

Solvation of the Glycyl Radical.

The effect of adding explicit water molecules to the neutral (N) and zwitterionic (Z) forms of the glycyl radical has been examined. The results show that a minimum of three water molecules is required to stabilize the Z radical as a local minimum, with an energy gap of 123 kJ mol-1 between the N and Z forms at this point, in favor of the N form. Increasing the number of water molecules to ∼20 leads to a converged Z-N energy difference of ∼50 kJ mol-1 still in favor of the N form, even though the radical is not considered fully solvated from a structural point of view. Thus, energetic convergence is determined mainly by solvation of the polar functional groups, and a complete coverage of the entire molecule is not necessary. Because aqueous closed-shell glycine exists as a zwitterion while aqueous glycyl radical prefers the neutral form, the conversion between the two necessitates a change along the hydrogen-abstraction reaction pathway. In this regard, the transition structure for α-hydrogen abstraction by the ·OH radical has greater resemblance to glycine than to the glycyl radical. Overall, the barrier for hydrogen abstraction from Z glycine is larger than that from the N isomer, and this might act to provide some protection against radical damage to the free amino acid in the (aqueous) biological environment.

Anaerobic Radical Enzymes for Biotechnology

AbstractEnzymes that proceed through radical intermediates have a rich chemistry that includes functionalization of otherwise unreactive carbon atoms, carbon‐skeleton rearrangements, aromatic reductions, and unusual eliminations. Especially under anaerobic conditions, organisms have developed a wide range of approaches for managing these transformations that can be exploited to generate new biological routes towards both bulk and specialty chemicals. These routes are often either much more direct or allow access to molecules that are inaccessible through standard (bio)chemical approaches. This review gives an overview of some of the key enzymes in this area: benzoyl‐CoA reductases effecting the enzymatic Birch reduction, ketyl radical dehydratases, coenzyme B12‐dependent enzymes, glycyl radical enzymes, and radical SAM (AdoMet radical) enzymes. These enzymes are discussed alongside biotechnological applications, highlighting the wide range of actual and potential uses. With the increased diversity in biotechnological approaches to obtaining these enzymes and information about them, even more of these enzymes can be expected to find application in industrial processes.

Anaerobic 4‐Hydroxyproline Metabolism by a Widespread Microbial Glycyl Radical Enzyme

Microbiome research has rapidly advanced through the use of high‐through put sequencing technologies, but the functions of most genes in sequence databases remain unknown. Glycyl radical enzymes represent an ancient protein super family, which is one of the most abundant protein families in the human gut microbiome. These enzymes use radical chemistry to catalyze a diverse set of chemically difficult transformations in metabolic pathways essential for anaerobic growth.Here, we describe the biochemical characterization of a new member of the glycyl radical enzyme superfamily, 4‐hydroxyproline dehydratase (HypD). This enzyme is responsible for the first step of anaerobic 4‐hydroxyproline (Hyp) metabolism. Hyp is the most abundant post‐translationally modified amino acid in the human proteome, with collagen as its principle source. It is also abundant in plant cell wall glycoproteins and functions as a site for O‐glycosylation. Hyp is present at significant levels in the human gut, deriving from both dietary sources and endogenous collagen turnover. Anaerobic Hyp metabolism had only been observed in Clostridiales as a part of Stickland fermentation in energy metabolism, but the enzymes and intermediates involved had not been identified until our work.We predicted the catalytic activity of HypD based on its sequence similarity to characterized glycyl radical enzymes and its genomic context in Clostridial species. Using purified enzymes, we demonstrated that HypD catalyzes the removal of water from Hyp to form pyrroline‐5‐carboxylate, a central intermediate in amino acid metabolism. As with all other glycyl radical enzymes, HypD activity requires post‐translational modification by a partner radical SAM enzyme to install a radical on a conserved glycine in the active site. We next examined the substrate scope and kinetics of HypD and demonstrated that it is stereospecific for trans‐4‐hydroxy‐L‐proline. Additional biochemical work has yielded insights into the radical‐based mechanism of this intriguing transformation. Our discovery of the biochemical basis for anaerobic Hyp metabolism has enabled us to uncover hundreds of strains with the potential for metabolizing Hyp. This under appreciated metabolism is encoded in species not known to ferment amino acids, where Hyp is likely used as a precursor for non‐Stickland pathways. In addition, HypD is widespread in common gut commensals as well as pathogens like Clostridiodes difficile, suggesting that it may represent a core function in the human gut microbiota. Furthermore, our work on the characterization of HypD has led us to new research directions to investigate related pathways in the gut microbiome.Support or Funding InformationFunded by Harvard University, a Packard Fellowship for Science and Engineering, and NSERC Postgraduate Scholarship‐Doctoral Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than frompetroleum.

Characterization of 1,2-Propanediol Dehydratases Reveals Distinct Mechanisms for B12-Dependent and Glycyl Radical Enzymes.

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of ( S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B12-dependent propanediol dehydratase (B12-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B12-PD. Here we report 18O labeling experiments that suggest PD and B12-PD employ distinct mechanisms. Unlike B12-PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1- gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

ABSTRACTThe discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.

Purification and Characterization of the Choline Trimethylamine-Lyase (CutC)-Activating Protein CutD.

Anaerobic choline deamination catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as a major route for trimethylamine (TMA) production within anaerobic environments, including the human gut. The association of this microbial metabolite and its downstream products with diseases such as atherosclerosis and chronic kidney disease has driven the need for a better molecular understanding of TMA-generating enzymes. Our previous work has shown that generating the critical, glycine-centered radical species on CutC requires posttranslational modification by an S-adenosyl-l-methionine (SAM)-dependent radical-activating protein (CutD) harboring an oxygen-sensitive [4Fe-4S] cofactor. In this chapter, we describe our strategy to heterologously express and purify Desulfovibrio alaskensis G20 CutD in Escherichia coli and reconstitute its iron-sulfur center under anaerobic conditions. In addition, we present the methods we have developed to characterize the activity of CutD and utilize this enzyme in conjunction with purified CutC to gain an unprecedented insight into the anaerobic C-N cleavage of choline.

The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity.

FocA is the archetype of the pentameric formate-nitrite transporter (FNT) superfamily of channels, members of which translocate small organic and inorganic anions across the cytoplasmic membrane of microorganisms. The N- and C-termini of each protomer are cytoplasmically oriented. A Y-L-R motif is found immediately after transmembrane helix 6 at the C-terminus of FNT proteins related to FocA, or those with a role in formate translocation. Previous in vivo studies had revealed that formate translocation through FocA was controlled by interaction with the formate-producing glycyl-radical enzyme pyruvate formate-lyase (PflB) or its structural and functional homolog, TdcE. In this study we analyzed the effect on in vivo formate export and import, as well as on the stability of the homopentamer in the membrane, of successively removing amino acid residues from the C-terminus of FocA. Removal of up to five amino acids was without consequence for either formate translocation or oligomer stability. Removal of a sixth residue (R280) prevented formate uptake by FocA in a strain lacking PflB and significantly reduced, but did not prevent, formate export. Sensitivity to the toxic formate analog hypophosphite, which is also transported into the cell by FocA, was also relieved. Circular dichroism spectroscopy and blue-native PAGE analysis revealed, however, that this variant had near identical secondary and quaternary structural properties to those of native FocA. Interaction with the glycyl radical enzyme, TdcE, was also unaffected by removal of the C-terminal 6 amino acid residues, indicating that impaired interaction with TdcE was not the reason for impaired formate translocation. Removal of a further residue (L279) severely restricted formate export, the stability of the protein and its ability to form homopentamers. Together, these studies revealed that the Y278-L279-R280 motif at the C-terminus is essential for bidirectional formate translocation by FocA, but that L279 is both necessary and sufficient for homopentamer integrity.

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M+ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[13C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster.

In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B12-dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B12-independent propanediol-utilizing BMC. Here we functionally and structurally characterize enzymes of the GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.

A prominent glycyl radical enzyme in human gut microbiomes metabolizes trans -4-hydroxy- l -proline

The human microbiome encodes vast numbers of uncharacterized enzymes, limiting our functional understanding of this community and its effects on host health and disease. By incorporating information about enzymatic chemistry into quantitative metagenomics, we determined the abundance and distribution of individual members of the glycyl radical enzyme superfamily among the microbiomes of healthy humans. We identified many uncharacterized family members, including a universally distributed enzyme that enables commensal gut microbes and human pathogens to dehydrate trans-4-hydroxy-l-proline, the product of the most abundant human posttranslational modification. This "chemically guided functional profiling" workflow can therefore use ecological context to facilitate the discovery of enzymes in microbial communities.

Cutting Choline with Radical Scissors

The human gut microbiome is the source of not only microbial diversity, but also of interesting chemical reactions and enzymology. An excellent example of this is CutC, an enzyme that makes trimethylamine (TMA). In this issue of Cell Chemical Biology, Bodea etal. (2016) show how CutC uses a glycyl radical to perform C-N bond cleavage needed for TMA production.

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production.

In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community.

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.

Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.

Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.

1,2-Propanediol Dehydration in Roseburia inulinivorans

Glycyl radical enzymes (GREs) represent a diverse superfamily of enzymes that utilize a radical mechanism to catalyze difficult, but often essential, chemical reactions. In this work we present the first biochemical and structural data for a GRE-type diol dehydratase from the organism Roseburia inulinivorans (RiDD). Despite high sequence (48% identity) and structural similarity to the GRE-type glycerol dehydratase from Clostridium butyricum, we demonstrate that the RiDD is in fact a diol dehydratase. In addition, the RiDD will utilize both (S)-1,2-propanediol and (R)-1,2-propanediol as a substrate, with an observed preference for the S enantiomer. Based on the new structural information we developed and successfully tested a hypothesis that explains the functional differences we observe.

Correction to Characterization of Choline Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical Enzymes

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to Characterization of Choline Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical EnzymesSmaranda Craciun, Jonathan A. Marks, and Emily P. Balskus*Cite this: ACS Chem. Biol. 2016, 11, 7, 2068Publication Date (Web):June 16, 2016Publication History Published online16 June 2016Published inissue 15 July 2016https://doi.org/10.1021/acschembio.6b00487Copyright © 2016 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views894Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (121 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Modeling of the Reaction Mechanism of Enzymatic Radical C-C Coupling by Benzylsuccinate Synthase.

Molecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical C–C coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. In this work docking calculations, classical molecular dynamics (MD) simulations, and DFT+D2 cluster modeling was employed to address the following questions: (i) What mechanistic details of the BSS reaction yield the most probable molecular model? (ii) What is the molecular basis of enantiospecificity of BSS? (iii) Is the proposed mechanism consistent with experimental observations, such as an inversion of the stereochemistry of the benzylic protons, syn addition of toluene to fumarate, exclusive production of (R)-benzylsuccinate as a product and a kinetic isotope effect (KIE) ranging between 2 and 4? The quantum mechanics (QM) modeling confirms that the previously proposed hypothetical mechanism is the most probable among several variants considered, although C–H activation and not C–C coupling turns out to be the rate limiting step. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro(R) orientation and reverse preference of benzyl radical attack on fumarate in pro(S) pathway which results with prohibitively high energy barrier of the radical quenching. Finally, the proposed mechanism agrees with most of the experimental observations, although the calculated intrinsic KIE from the model (6.5) is still higher than the experimentally observed values (4.0) which suggests that both C–H activation and radical quenching may jointly be involved in the kinetic control of the reaction.

Snapshots Of Benzylsuccinate Synthase: Getting A Handle On Toluene Degradation

Anaerobic bacteria play a critical role in bioremediation of oil spills and industrial pollution. The first step in the anaerobic degradation of toluene, a major component of gasoline, is the addition of toluene to fumarate to yield benzylsuccinate. The enzyme responsible is benzylsuccinate synthase (BSS), a member of glycyl radical enzyme family, which uses free radical chemistry to break and form C‐H and C‐C bonds. Here, crystallographic snapshots of BSS are presented that allow us to consider how a glycyl radical enzyme can control substrate specificity to afford activation of the otherwise inert toluene molecule.Support or Funding InformationThis work was supported in part by the National Science Foundation Graduate Research Fellowship under Grant No. 0645960 (M.A.F.). C.L.D is a Howard Hughes Medical Institute (HHMI) Investigator.

Metabolism of Hydrocarbons in n-Alkane-Utilizing Anaerobic Bacteria

The glycyl radical enzyme-catalyzed addition of n-alkanes to fumarate creates a C-C-bond between two concomitantly formed stereogenic carbon centers. The configurations of the two diastereoisomers of the product resulting from n-hexane activation by the n-alkane-utilizing denitrifying bacterium strain HxN1, i.e. (1-methylpentyl)succinate, were assigned as (2S,1′R) and (2R,1′R). Experiments with stereospecifically deuterated n-(2,5-²H₂)hexanes revealed that exclusively the pro-S hydrogen atom is abstracted from C2 of the n-alkane by the enzyme and later transferred back to C3 of the alkylsuccinate formed. These results indicate that the alkylsuccinate-forming reaction proceeds with an inversion of configuration at the carbon atom (C2) of the n-alkane forming the new C-C-bond, and thus stereochemically resembles a S_N2-type reaction. Therefore, the reaction may occur in a concerted manner, which may avoid the highly energetic hex-2-yl radical as an intermediate. The reaction is associated with a significant primary kinetic isotope effect (kH/kD ≥3) for hydrogen, indicating that the homolytic C-H-bond cleavage is involved in the first irreversible step of the reaction mechanism. The (1-methylalkyl)succinate synthases of n-alkane-utilizing anaerobic bacteria apparently have very broad substrate ranges enabling them to activate not only aliphatic but also alkyl-aromatic hydrocarbons. Thus, two denitrifiers and one sulfate reducer were shown to convert the nongrowth substrate toluene to benzylsuccinate and further to the dead-end product benzoyl-CoA. For this purpose, however, the modified β-oxidation pathway known from alkylbenzene-utilizing bacteria was not employed, but rather the pathway used for n-alkane degradation involving CoA ligation, carbon skeleton rearrangement and decarboxylation. Furthermore, various n-alkane- and alkylbenzene-utilizing denitrifiers and sulfate reducers were found to be capable of forming benzyl alcohols from diverse alkylbenzenes, putatively via dehydrogenases. The thermophilic sulfate reducer strain TD3 forms n-alkylsuccinates during growth with n-alkanes or crude oil, which, based on the observed patterns of homologs, do not derive from a terminal activation of n-alkanes.