Genes encoding the candidate enzyme for anaerobic activation of n‐alkanes in the denitrifying bacterium, strain HxN1

Abstract

Strain HxN1, a member of the Betaproteobacteria, can grow anaerobically by denitrification with n-alkanes. n-Alkanes are apparently activated by subterminal carbon addition to fumarate yielding (1-methylalkyl)succinates, the postulated enzyme being (1-methylalkyl)succinate synthase (Mas). Genes encoding this enzyme (mas) were searched for via proteins that were specifically formed in n-hexane-grown cells (in comparison with caproate-grown cells), as revealed by two-dimensional gel electrophoresis. Partial amino acid sequencing and subsequent probe development for hybridization of restricted DNA led to the identification of a gene cluster. Deduced proteins are similar to the subunits of benzylsuccinate synthase (Bss), the toluene-activating enzyme in other anaerobic bacteria and its activase. The tentative (1-methylalkyl)succinate synthase is presumably a heterotrimer (MasDEC) which, like benzylsuccinate synthase, contains a motif (in MasD, the large subunit) characteristic of glycyl radical-bearing sites. Based on amino acid sequence comparison, the tentative (1-methylalkyl)succinate synthase branches outside of the phylogenetic cluster of benzylsuccinate synthases from different organisms and represents a separate line of descent within glycyl radical enzymes. n-Hexane-induced co-transcription of the mas genes and additional genes of an apparent operon was demonstrated by Northern hybridization experiments.

Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47

“…The ion selection threshold for MS/MS was 500 counts, and an activation Q-value (effective collision energy) of 0. 25 and an activation time of 30 ms were also applied for MS/MS.…”

“…The Bss enzyme is a well-investigated close homolog of Nms, catalyzing fumarate addition in the initial reaction of anaerobic toluene degradation (34,40). Based on findings from comparative sequence studies, glycine radical-catalyzed fumarate addition has been shown to be a widely distributed initial reaction mechanism for anaerobic hydrocarbon degradation involving toluene and 2-methylnaphthalene, n-alkanes (12,13,25,51), m-xylene (33), m-and p-cresols (9), and ethylbenzene (32).…”

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

“…These genes may well be active in the degradation of hydrocarbons other than toluene, which can also involve fumarate addition (Callaghan et al, 2008;Grundmann et al, 2008;Musat et al, 2009). Alternatively, it could indicate that intrinsic toluene-degrading microbial populations at the site have changed between the two time points of sampling (August 2004 andDecember 2006), which is supported by the fact that in 2004 we did not detect any 'F2'-lineage bssA genes at the site, whereas direct bssA-targeted fingerprinting of DNA from our SIP inoculum, taken from the same well as in 2004, did.…”

Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation

“…1B) (11,12). Additionally, alkyl-SSs, which function on alkyl chains including n-hexane, have also been discovered (13,14), although there is less known about the substrate specificity and mechanism of these enzymes. Together these enzymes form a large family of X-succinate synthases (XSSs) responsible for functionalization of a wide variety of hydrocarbon substrates, and the range of substrates is still growing.…”