Identification of the Final Two Genes Functioning in Methanofuran Biosynthesis in Methanocaldococcus jannaschii

Abstract

All methanofuran structural variants contain a basic core structure of 4-[N-(␥-L-glutamyl)-p-(␤-aminoethyl)phenoxymethyl]-(aminomethyl)furan (APMF-Glu) but have different side chains depending on the source organism. Recently, we identified four genes (MfnA, MfnB, MfnC, and MfnD) that are responsible for the biosynthesis of the methanofuran precursor ␥-glutamyltyramine and 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P) from tyrosine, glutamate, glyceraldehyde-3-P, and alanine in Methanocaldococcus jannaschii. How ␥-glutamyltyramine and F1-P couple together to form the core structure of methanofuran was previously unknown. Here, we report the identification of two enzymes encoded by the genes mj0458 and mj0840 that catalyze the formation of F1-PP from ATP and F1-P and the condensation of F1-PP with ␥-glutamyltyramine, respectively, to form APMF-Glu. We have annotated these enzymes as MfnE and MfnF, respectively, representing the fifth and sixth enzymes in the methanofuran biosynthetic pathway to be identified. Although MfnE was previously reported as an archaeal adenylate kinase, our present results show that MfnE is a promiscuous enzyme and that its possible physiological role is to produce F1-PP. Unlike other enzymes catalyzing coupling reactions involving pyrophosphate as the leaving group, MfnF exhibits a distinctive ␣/␤ twolayer sandwich structure. By comparing MfnF with thiamine synthase and dihydropteroate synthase, a substitution nucleophilic unimolecular (S N -1) reaction mechanism is proposed for MfnF. With the identification of MfnE and MfnF, the biosynthetic pathway for the methanofuran core structure APMF-Glu is complete. IMPORTANCEThis work describes the identification of the final two enzymes responsible for catalyzing the biosynthesis of the core structure of methanofuran. The gene products of mj0458 and mj0840 catalyze the formation of F1-PP and the coupling of F1-PP with ␥-glutamyltyramine, respectively, to form APMF-Glu. Although the chemistry of such a coupling reaction is widespread in biochemistry, we provide here the first evidence that such a mechanism is used in methanofuran biosynthesis. MfnF belongs to the hydantoinase A family (PF01968) and exhibits a unique ␣/␤ two-layer sandwich structure that is different from the enzymes catalyzing similar reactions. Our results show that MfnF catalyzes the formation of an ether bond during methanofuran biosynthesis. Therefore, this work further expands the functionality of this enzyme family. Methanofuran is the first in a series of coenzymes involved in the biochemical reduction of carbon dioxide to methane (1-3). This process, known as methanogenesis, is carried out only by the methanogenic archaea, which produce more than 400 million tons of methane each year as an essential part of the global carbon cycle (4). Methanofuran is the initial C 1 acceptor molecule involved in the first two-electron reduction of carbon dioxide to produce the formamide derivative of methanofuran, where the formate is attached to the amino group of methanofu...

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

“…This change allows comparison of differential expression of tRNA genes with metabolic flux. Fourth, the biosynthesis pathway for methanofuran was updated based on recent characterization of the five mfn genes [52–55]. Methanofuran is a component in the methyl-oxidation branch of methanogenesis playing a role in a key redox step inter-converting a formyl group and CO 2 and is required for all modes of methanogenic growth [37, 56].…”

Industrial production, application, microbial biosynthesis and degradation of furanic compound, hydroxymethylfurfural (HMF)

“…Another example is a novel enzyme, 4-(hydroxymethyl)-2-furancarboxyaldehyde-phosphate synthase (MfnB) [34],[35]. MfnB catalyzes the formation of 4-hydroxymethylfurfural phosphate (4-HMF-P) from two molecules of D-glyceraldehyde-3phosphate (GA-3P) in the biosynthetic pathway to the furan moiety of the coenzyme methanofuran found in methanogens (Figure 3) [36]. The crystal structure of MfnB exhibits a typical α/β TIM barrel fold, which is commonly seen within Class I aldolases [37]–[44].…”

Genome-Scale Metabolic Modeling of Archaea Lends Insight into Diversity of Metabolic Function

“…The biomass, GAM, and NGAM requirements remained the same as those from i MB745. In i ST807, we updated i MB745 by revising the methanofuran biosynthesis pathway with the most recent experimental data from M. jannaschii [84–87], adding 13 new reactions and 62 new genes, and revising the biomass reaction to utilize charged tRNAs instead of free amino acids. Among the new additions are reactions to enable pyrrolysine biosynthesis during methylamine growth, methyl-3-mercaptopropionate metabolism, and o-phosphoserine conversion to cysteine after aminoacylation.…”