A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate

Abstract

Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase (Mch) and coenzyme F420 -dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyse the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent Km for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO₂in ANME involves the same enzymes and coenzymes as CO₂reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1 were found to be dependent on the presence of 0.5-1.0 M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…In support of this, genes encoding homologs of Mtd and Mer have been found in some reconstructed ANME metagenomes (21,137,215). Heterologously expressed Mtd from an ANME-1 archaeon catalyzed the same reaction as Mtd from methanogens, with similar catalytic specificity and cofactor dependence (214). In addition to F 420 -dependent enzymes, NAD(P)-dependent methylenetetrahydromethanopterin dehydrogenases have been characterized that have central roles in the formaldehyde assimilation pathways of aerobic methylotrophic bacteria (301,302).…”

“…A range of genomic and biochemical evidence suggests that these archaea predominantly grow through a reverse methanogenesis pathway (similar to the methylotrophic pathway; Fig. 7), through which F 420 H 2 is generated via the Mer and Mtd reactions (137,(212)(213)(214)(215). The F 420 H 2 that is produced from this pathway is proposed to be reoxidized by the protontranslocating Fqo complex, with sulfate or nitrate serving as the terminal respiratory electron acceptor (21,215).…”

Catabolic Pathways and Enzymes Involved in Anaerobic Methane Oxidation

“…The genes from the metagenom sequences of ANME-1 were heterologously overexpressed and the enzymes Mtd, Mch and Ftr were purified to homogeneity by column chromatography without tags (Kojima et al 2014). The three enzymes revealed high activity at 10 °C, which is near the physiological temperature of the ANME-1 archaea harvested.…”

Methane oxidation by anaerobic archaea for conversion to liquid fuels

“…A homolog to Mcr was purified and characterized from anoxic sediments and shown to bind coenzyme M and coenzyme B [80] and initiate the first step of reverse methanogenesis in the presence of sulfate [45,52]. Homologs from ANME-1 for three other methanogenesis enzymes, formyl-MFR:H 4 MPT formyltransferase (Ftr), methenyl-H 4 MPT cyclohydrolase (Mch), and F 420 -dependent methylene-H 4 MPT dehydrogenase (Mtd) were synthesized and expressed in Escherichia coli [44]. In the presence of the corresponding coenzymes, the purified enzymes showed activity for their native substrates [44].…”

“…Homologs from ANME-1 for three other methanogenesis enzymes, formyl-MFR:H 4 MPT formyltransferase (Ftr), methenyl-H 4 MPT cyclohydrolase (Mch), and F 420 -dependent methylene-H 4 MPT dehydrogenase (Mtd) were synthesized and expressed in Escherichia coli [44]. In the presence of the corresponding coenzymes, the purified enzymes showed activity for their native substrates [44]. The presence of these methanogenesis pathway genes in ANME organisms along with the ability of Mcr to catalyze the first step in the reverse methanogenic pathway [45,52] provides support for the activity of reverse methanogenesis in ANME.…”