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Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain.

Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain.

Authors :
Hemmann JL
Brühwiler MR
Bortfeld-Miller M
Vorholt JA
Source :
The Journal of biological chemistry [J Biol Chem] 2021 Jan-Jun; Vol. 296, pp. 100682. Date of Electronic Publication: 2021 May 01.
Publication Year :
2021

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.<br />Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.<br /> (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Details

Language :
English
ISSN :
1083-351X
Volume :
296
Database :
MEDLINE
Journal :
The Journal of biological chemistry
Publication Type :
Academic Journal
Accession number :
33894199
Full Text :
https://doi.org/10.1016/j.jbc.2021.100682