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Here, the biochemical and structural properties of MTHFR from a virulent strain of <jats:italic>N. meningitidis<\/jats:italic> serogroup B (<jats:italic>Nm<\/jats:italic>MTHFR) were characterized. Unlike other orthologs, <jats:italic>Nm<\/jats:italic>MTHFR functions as a unique homohexamer, composed of three homo\u2010dimerization partners, as shown in our 2.7\u2009\u00c5 resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH<jats:sub>2<\/jats:sub>\u2010H<jats:sub>4<\/jats:sub>folate substrate. <jats:italic>Nm<\/jats:italic>MTHFR utilized NADH (<jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub>\u2009=\u200944\u2009\u03bcM) as an electron donor in the NAD(P)H\u2010CH<jats:sub>2<\/jats:sub>\u2010H<jats:sub>4<\/jats:sub>folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. <jats:italic>In silico<\/jats:italic> analysis and mutagenesis studies highlighted the significant difference in orientation of helix \u03b17A (Phe215\u2013Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix \u03b17A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH<jats:sub>2<\/jats:sub>\u2010H<jats:sub>4<\/jats:sub>folate substrate to bind. The unique topology of its NADH and CH<jats:sub>2<\/jats:sub>\u2010H<jats:sub>4<\/jats:sub>folate binding pockets makes <jats:italic>Nm<\/jats:italic>MTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off\u2010target side effects.<\/jats:p>","DOI":"10.1002\/pro.4654","type":"journal-article","created":{"date-parts":[[2023,5,11]],"date-time":"2023-05-11T06:14:50Z","timestamp":1683785690000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase"],"prefix":"10.1002","volume":"32","author":[{"given":"Wanita","family":"Pantong","sequence":"first","affiliation":[{"name":"Division of Health and Applied Sciences, Faculty of Science Prince of Songkla University  Songkhla Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jordan 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