Abstract
Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography-mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.