Degradation Pathways

AnHyDeg: A Curated Database of Anaerobic Hydrocarbon Degradation Genes

Amy V. Callaghan^ and Boris Wawrik*

^* Corresponding Author

University of Oklahoma

Department of Microbiology and Plant Biology, 770 Van Vleet Oval, Norman OK 73019

Please cite as follows:

Callaghan, A.V., and B. Wawrik. 2016. AnHyDeg: A Curated Database of Anaerobic Hydrocarbon Degradation Genes. https://github.com/AnaerobesRock/AnHyDeg. DOI:

Anaerobic transformation of hydrocarbons by microorganisms contributes to biogeochemical cycling in anoxic ecosystems and to the bioremediation of contaminated environments. During the past twenty-five years, a great deal has been learned about anaerobic biodegradation processes and the genetic machinery involved in the requisite biochemical pathways. Functional genes are frequently used as biomarkers in environmental assessments of anaerobic hydrocarbon metabolism, and metagenomic techniques have vastly enhanced our ability to interrogate consortia and environmental systems for these gene markers. However, the accrued lack of manual curation of ‘omic’ datasets has led to the misannotation of these gene and protein sequences in several public databases. The GitHub database herein was designed to provide a starting point for building a highly curated database of sequences associated with anaerobic hydrocarbon degradation processes. The sequences included here are for genes/proteins that are mainly involved in the anaerobic activation of hydrocarbons and that have been described for well characterized isolates or consortia. This database will continue to be updated to include information for additional substrates and sequences from other isolates/consortia. The long-term objective will be to include data for downstream pathway steps.

NOTE: The following are only brief summaries of proposed activation steps for different hydrocarbon classes. However, we encourage the users of this GitHub entry to refer to a recent 2016 issue of the Journal of Molecular Microbiology and Biotechnology focusing on anaerobic hydrocarbon degradation [Volume 26(1-3), pages 1-244] for the complete descriptions of the requisite pathways and proteins.

Degradation Pathways

Alkanes
[BTEX & Related Compounds] (https://github.com/OUGenomics/AnaerobicHydrocarbonDegradationGenes/blob/master/README.md#btex--related-compounds)
[Naphthalene and other PAHs] (https://github.com/OUGenomics/AnaerobicHydrocarbonDegradationGenes/blob/master/README.md#naphthalene-and-other-pahs)
[Hydroxylated Hydrocarbons] (https://github.com/OUGenomics/AnaerobicHydrocarbonDegradationGenes/blob/master/README.md#hydroxylated-hydrocarbons)
- [Phenol] (https://github.com/OUGenomics/AnaerobicHydrocarbonDegradationGenes/blob/master/README.md#phenol)
- [p-Cresol] (https://github.com/OUGenomics/AnaerobicHydrocarbonDegradationGenes/blob/master/README.md#p-cresol)

##Non-Methane Linear Alkanes and Branched Alkanes

To date, there is one, well-characterized, oxygen-independent mechanism of anaerobic alkane activation, which is alkane addition to fumarate. This process has been described for a diverse range of bacteria utilizing linear and branched alkanes (for review see (Wilkes et al., 2016)). The mechanism is catalyzed by the glycyl radical enzyme, alkylsuccinate synthase (ASS) (Callaghan et al., 2008a; Herath et al., 2016) (also known as methylalkylsuccinate synthase, MAS) (Grundmann et al., 2008; Webner, 2012). Typically, alkane addition occurs at the C2 carbon to form 2-methylalkylsuccinic acids (for review see (Callaghan, 2013)). However some studies have shown terminal (C1) (Kniemeyer et al., 2007; Jaekel et al., 2014) or C3 (Rabus et al., 2001) addition to fumarate. Interestingly, a recent study of thermophilic strain TD3 suggests that in some systems the detection of n-alkylsuccinates may in fact be indicative of transformation of short-chain fatty acids via carboxylation and C-skeleton rearrangement rather than terminal addition of alkanes to fumarate (Jarling et al., 2015).

Anaerobic hydroxylation has also been proposed as a putative mechanism for anaerobic alkane transformation. Initial metabolite profiling studies of Desulfococcus oleovorans Hxd3 suggested that the activation step may be direct carboxylation at C3, followed by oxidation releasing the terminal and subterminal carbons (So et al., 2003), resulting in a fatty acid that is one carbon shorter than the parent alkane. However, the requisite metabolite, 2-ethylpentadecanoic acid, was never identified in Hxd3 cultures or others that had demonstrated similar fatty acid profiles (Callaghan et al., 2006; Callaghan et al., 2009). Subsequently, a genome survey of Hxd3 revealed a protein complex related to ethylbenzene dehydrogenase (Callaghan et al., 2008b), and it has been hypothesized that this protein may be a putative alkane C2-methylene hydroxylase complex (AHY), wherein anaerobic hydroxylation at C2, followed by formation of a subsequent ketone and carboxylation at C3 may occur, with oxidation to produce a fatty acid that is one carbon shorter than the parent alkane (Heider and Schühle, 2013). Although not substantiated by metabolite analysis, substrate-dependent induction of the respective gene during growth on alkanes suggests that this mechanism may occur (Sunwoldt and Heider, unpublished data).

####Alkylsuccinate Synthase (ASS) / (Methyl)alkylsuccinate Synthase (MAS)

ASS/MAS	GenBank Annotation Info
Alpha subunit	Glycyl radical enzyme, PFL2/glycerol dehydratase family; TIGR01774
Beta subunit
Gamma subunit	BssC/TutF protein-like protein; pfam08201
MasE and MasE-like proteins	Includes uncharacterized proteins similar to MasE
Activase	Radical SAM superfamily. cl18962 TIGR04003

####Putative Alkane C2-Methylene Hydroxylase

AHY	GenBank Annotation Info
Alpha subunit	DMSO reductase family type II enzyme, molybdopterin subunit; TIGR03479
Beta subunit	Respiratory nitrate reductase / 4Fe-4S ferredoxin iron-sulfur binding domain protein; TIGR03478
Gamma subunit	Heme-binding
Delta subunit	Chaperone

##BTEX & Related Compounds

###Benzene

Several mechanisms of anaerobic benzene activation have been proposed, including hydroxylation, methylation followed by toluene addition to fumarate, and carboxylation (for review see (Meckenstock and Mouttaki, 2011)). The carboxylation of benzene appears to be catalyzed by a putative benzene carboxylase (ABC). The number of subunits is still debated. To date, subunits AbcA and AbcD were detected in an anaerobic, iron-reducing, enrichment culture (Abu Laban et al., 2010), whereas only AbcA was detected in the genome of benzene-degrading archaeon, Ferroglobus placidus (Holmes et al., 2011).

#####Putative Benzene Carboxylase

ABC	GenBank Annotation Info
Putative benzene carboxylase	3-Octaprenyl-4-hydroxybenzoate carboxy-lyase; cl00311
Gamma subunit	Phenylphosphate carboxylase, gamma subunit; TIGR02725

Toluene

The anaerobic activation of toluene proceeds by methyl addition to fumarate to yield benzylsuccinate (Evans et al., 1992; Biegert et al., 1996; Beller and Spormann, 1997a, b). This reaction is catalyzed by the glycyl radical enzyme, benzylsuccinate synthase (BSS) (Leuthner et al., 1998; Beller and Spormann, 1999). Note – In Thauera aromatica T1, this enzyme is referred to as TUT (toluene-utilizing) (Coschigano et al., 1998).

###Xylene Isomers

The isomers of xylene (o-, m-, and p-xylene) are activated under anaerobic conditions via addition of a methyl group to fumarate to yield 2-methylbenzylsuccinate, 3-methylbenzylsuccinate, and 4-methylbenzylsuccinate, respectively (Evans et al., 1992; Beller and Spormann, 1997b; Krieger et al., 1999; Verfürth et al., 2004; Morasch and Meckenstock, 2005). To date, these reactions are catalyzed by BSS orthologs in different species (for review see (Heider et al., 2016b).

#####Benzylsuccinate Synthase

BSS	GenBank Annotation Info
Alpha subunit	Glycyl radical enzyme, PFL2/glycerol dehydratase family; TIGR01774
Beta subunit
Gamma subunit	BssC/TutF protein; pfam08201
Activase	Radical SAM superfamily. cl18962

###Ethylbenzene

There are two mechanisms of anaerobic activation of ethylbenzene : 1) anaerobic hydroxylation via ethylbenzene dehydrogenase (EBDH), wherein water serves as the source of the hydroxyl group (for review see (Heider et al., 2016) and addition to fumarate (Kniemeyer et al., 2003), presumably by a homolog of benzylsuccinate synthase. Of the two enzymes, EBDH is well described and has been identified and/or purified in betaproteobacterial strains EB1 (Ball et al., 1996; Johnson et al., 2001), EbN1 (Rabus and Widdel, 1995; Kniemeyer and Heider, 2001b), and PbN1 (Rabus and Widdel, 1995). Hydroxylation of ethylbenzene yields (S)-1-phenylethanol, which is further oxidized to acetophenone by (S)-1-phenylethanol dehydrogenase (PEDH) (Kniemeyer and Heider, 2001a; Höffken et al., 2006); acetophenone is then carboxylated to benzoylacetate by acetophenone carboxylase (ACP) (Jobst et al., 2010).

#####Ethylbenzene Dehydrogenase

EBDH	GenBank Annotation Info
Alpha subunit	DMSO reductase family type II enzyme, molybdopterin subunit; TIGR03479
Beta subunit	Respiratory nitrate reductase / 4Fe-4S ferredoxin iron-sulfur binding domain protein
Gamma subunit	Heme-binding
Delta subunit	Chaperone

#####Phenylethanol Dehydrogenase

PEDH	GenBank Annotation Info
Phenylethanol dehydrogenase	3-Ketoacyl-(acyl-carrier-protein) reductase; PRK0555

#####Acetophenone Carboxylase

APC	GenBank Annotation Info
Alpha subunit	N-Methylhydantoinase A/oxoprolinase/acetone carboxylase, beta subunit [Amino acid transport and metabolism, aecondary metabolites biosynthesis, transport and catabolism]; COG0145
Beta subunit	Acetone carboxylase gamma subunit; pfam08882
Gamma subunit	N-Methylhydantoinase A/oxoprolinase/acetone carboxylase, beta subunit [Amino acid transport and metabolism, aecondary metabolites biosynthesis, transport and catabolism]; COG0145
Delta subunit	Hydantoinase B/oxoprolinase; cl19816
Epsilon subunit	The URO-D_CIMS_like protein; cl00464

###p-Cymene (4-Isopropyltoluene)

Anaerobic p-cymene degradation proceeds either by: 1) hydroxylation of the benzylic methyl group via cymene dehydrogenase (CDH) (Strijkstra et al., 2014), or 2) addition of the benzylic methyl group to fumarate (Harms et al., 1999; Strijkstra et al., 2014), which is catalyzed by (4-isopropylbenzyl)succinate synthase (IBS) (Strijkstra et al., 2014). These enzymes were identified in denitrifying organisms 'Aromatoleum’ aromaticum pCyN1 and Thauera sp. Strain pCyN2, respectively (for review see (Rabus et al., 2016)

#####p-Cymene Dehydrogenase

CMD	GenBank Annotation Info
Alpha subunit	DMSO reductase family type II enzyme, molybdopterin subunit; TIGR03479
Beta subunit	DMSO reductase family type II enzyme, iron-sulfur subunit; TIGR03478
Gamma subunit	Domon-like ligand-binding domains; cl14783
Chaperone assembly protein	Nitrate reductase delta subunit; cl00958

#####(4-Isopropylbenzyl)succinate Synthase

IBS	GenBank Annotation Info
Alpha subunit	Glycyl radical enzyme, PFL2/glycerol dehydratase family; TIGR01774
Beta subunit
Gamma subunit	BssC/TutF protein-like protein; pfam08201
Activase	Radical SAM superfamily. cl18962 TIGR04003

##Naphthalene and other PAHs

To date, there are two, well-described mechanisms of anaerobic naphthalene activation: 1) direct carboxylation (Zhang and Young, 1997; Meckenstock et al., 2000), catalyzed by naphthalene carboxylase (DiDonato et al., 2010; Bergmann et al., 2011a; Bergmann et al., 2011b; Mouttaki et al., 2012) and 2) and methylation followed by fumarate addition (Annweiler et al., 2000; Annweiler et al., 2001, 2002; Safinowski and Meckenstock, 2004; Safinowski et al., 2006; Safinowski and Meckenstock, 2006), the latter being catalyzed by the glycyl radical enzyme, naphthyl-2-methylsuccinate synthase (NMS) (Annweiler et al., 2000; Safinowski and Meckenstock, 2004, 2006; Selesi et al., 2010). Although most studies addressing anaerobic PAH degradation have focused on naphthalene, there is evidence that the above mechanisms may be applicable to other PAHs such as 2-methylnaphthalene (Sullivan et al., 2001; Musat et al., 2009), 1-methylnaphthalene (Safinowski et al., 2006; Musat et al., 2009), phenanthrene (Zhang and Young, 1997; Davidova et al., 2007), biphenyl (Selesi and Meckenstock, 2009), and acenaphthene (Safinowski et al., 2006; Jobelius et al., 2011; Morasch et al., 2011).

NOTE: An initial survey of the Desulfobacterium sp. N47 revealed four putative beta subunits of naphthalene carboxylase (Locus tags: N47_K27500, N47_K27480, N47_K27460, and N47_K27390) (Bergmann et al., 2011a), three of which were found to be expressed when N47 was grown on naphthalene (N47_K27500, N47_K27480, N47_K27460) (Bergmann et al., 2011b). The latter are included in this database. Similarly, a gene cluster homologous to that in N47 was described for Deltaproteobacterium Naph S2 (DiDonato et al., 2010).

#####2-Napthylmethylsuccinate Synthase

NMS	GenBank Annotation Info
Alpha subunit	Glycyl radical enzyme, PFL2/glycerol dehydratase family; TIGR01774
Beta subunit
Gamma subunit	BssC/TutF protein; pfam08201
Activase	Radical SAM superfamily; 4Fe-4S dicluster domain; pfam13183

#####Naphthalene Carboxylase

Nap Carboxylase	GenBank Annotation Info
Alpha subunit	match to protein family HMM PF01977; match to protein family HMM TIGR00148
Beta subunit	hypothetical protein; match to protein family HMM PF01977

##Hydroxylated Hydrocarbons

###Phenol

Anaerobic phenol degradation proceeds via activation to phenylphosphate by phenylphosphate synthase, and then carboxylation of phenylphosphate to form 4-hydroxybenzoate by phenylphosphate carboxylase (Lack et al., 1991; Lack and Fuchs, 1992, 1994; Schühle and Fuchs, 2004; Wöhlbrand et al., 2007; Schleinitz et al., 2009; Abu Laban et al., 2010).

#####Phenylphosphate Synthase

PPS	GenBank Annotation Info
Alpha subunit	PEP-utilizing enzyme, mobile domain; pfam00391
Beta subunit	Pyruvate phosphate dikinase, PEP/pyruvate binding domain; pfam01326

#####Phenylphosphate Carboxylase

PPC	GenBank Annotation Info
Alpha subunit	Phenylphosphate carboxylase, alpha subunit; TIGR02723
Beta subunit	3-Octaprenyl-4-hydroxybenzoate carboxy-lyase; cl00311
Gamma subunit	Haloacid dehalogenase-like hydrolase; pfam00702
Delta subunit	Phenylphosphate carboxylase, gamma subunit; TIGR02725

###p-Cresol

Anaerobic biodegradation of p-cresol proceeds via: 1) methyl addition to fumarate by hydroxybenzylsuccinate synthase (HBS) (Müller et al., 2001; Wöhlbrand et al., 2013), or 2) anaerobic hydroxylation by p-cresol methylhydroxylase (PCMH) (Keat and Hopper, 1978; Hopper et al., 1991; Peters et al., 2007).

#####Hydroxybenzylsuccinate Synthase

HBS	GenBank Annotation Info
Alpha subunit	Glycyl radical enzyme, PFL2/glycerol dehydratase family; TIGR01774
Beta subunit
Gamma subunit	BssC/TutF protein-like protein; pfam08201
Activase	Radical SAM superfamily. cl18962 TIGR04003

#####p-Cresol Methylhydroxylase

PCMH	GenBank Annotation Info
PcmI/J Alpha subunit isoforms	FAD linked oxidases, C-terminal domain; pfam02913; COG0277
PcmG cytochrome C Beta Subunit	Cytochrome c, mono- and diheme variants; COG2010; pfam13442

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