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Protein Sci ; 32(6): e4654, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37165541

RESUMO

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 Å 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 -H4 folate substrate. NmMTHFR utilized NADH (Km = 44 µM) as an electron donor in the NAD(P)H-CH2 -H4 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 α7A (Phe215-Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A 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 -H4 folate substrate to bind. The unique topology of its NADH and CH2 -H4 folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2) , Neisseria meningitidis , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , NAD/química , NADP , Modelos Moleculares , Ácido Fólico/química , Ácido Fólico/metabolismo , Neisseria meningitidis/metabolismo , Adenina
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