Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase
Document Type
Article
Publication Date
6-1-2023
Abstract
Methylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis, a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B (NmMTHFR) were characterized. Unlike other orthologs, NmMTHFR functions as a unique homohexamer, composed of three homo-dimerization partners, as shown in our 2.7 angstrom 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 CH2-H(4)folate substrate. NmMTHFR utilized NADH (K-m = 44 mu M) as an electron donor in the NAD(P)H-CH2-H(4)folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix alpha 7A (Phe215-Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix alpha 7A 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 CH2-H(4)folate substrate to bind. The unique topology of its NADH and CH2-H(4)folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects.
Keywords
Folate cycle, Methionine biosynthesis, Methylenetetrahydrofolate reductase, Neisseria meningitidis, X-ray crystal structure
Divisions
fac_med
Funders
Prince of Songkla University [Grant No: 22393-SCI6202127N],Prince of Songkla University, Thailand,Office of the Higher Education Commission of Thailand,Mahidol University,National Research Council of Thailand (NRCT) Grant [Grant Ni: NRCT5-RSA63012-01],Ministry of Higher Education, Malaysia - Fundamental Research Grant [Grant No: FRGS/1/2019/SKK08/UM/02/16 ],Ministry of Education, Malaysia
Publication Title
Protein Science
Volume
32
Issue
6
Publisher
Wiley
Publisher Location
111 RIVER ST, HOBOKEN 07030-5774, NJ USA