Abstract
All methanofuran structural variants contain a basic core structure of 4-[N-(γ-l-glutamyl)-p-(β-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 γ-glutamyltyramine and 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P) from tyrosine, glutamate, glyceraldehyde-3-P, and alanine in Methanocaldococcus jannaschii. How γ-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 γ-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 α/β two-layer sandwich structure. By comparing MfnF with thiamine synthase and dihydropteroate synthase, a substitution nucleophilic unimolecular (SN-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. IMPORTANCE: This 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 γ-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 α/β 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.