Inhibition of 5,10-methenyltetrahydrofolate synthetase

ArticleinArchives of Biochemistry and Biophysics 458(2):194-201 · March 2007with18 Reads
DOI: 10.1016/j.abb.2006.12.023 · Source: PubMed
Abstract
The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.
    • "The catalytic inactivation may result from a conformational change which accompanies dissociation. Even though the binding experiments showed that there are four sites for each substrate per mole of tetramer, the binding sites may not be intrinsic to each monomer383940414243. There are two possible reasons; one or more of the substrate sites may be formed as a result of conformational changes which are caused by the protein-protein interactions responsible for the association of monomers, or one or more of the substrate sites may possibly be located at the site of interaction between subunits and that the substrate site was composed of segments of polypeptide chains from two distinct subunits. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: The objective was to design an enzyme construct with diverse function from urease sequences of haloarchaean, Haloarcula marismortui ATCC 43049 based on its conserved domain consisting metal-binding region and active sites. Methods: Complete urease sequences of haloarchaea were retrieved from National Center for Biotechnology Information and then homology models generated, and validated. The best protein models were selected for docking with respective substrates using Ligand Fit program. The lowest energetic conformers were generated from these protein models by molecular dynamics methods. Urease construct-substrate complex was chosen based on the mode of catalysis, types of molecular interactions, and binding energy. Results: The resulted construct has a monomeric structure consisting of 3 helixes and 6 turns with 97 amino acids in length. The side chains of Asp49, Gly50 and Gln51 were predicted as functional residues in this construct. Urease construct was predicted to show catalytic function as similar to aliphatic nitrile hydradase and acrylamide hydro-lyase. Binding affinity of construct was more significant, which was better than to native urease. Urease construct was showed high binding affinity with semicarbazide and acrylamide wherein it has formed favorable hydrogen bonds. Conclusion: Substrate-binding region and active sites in the conserved domain of haloarchaean ureases are evolutionarily conserved at sequence as well as structural level. Substrate docking study supports the strong molecular interactions between construct and relative substrates. Thus, the present approach provides an insight to design urease construct with diverged catalytic function.
    Full-text · Article · Jul 2012
    • "The catalytic inactivation may result from a conformational change which accompanies dissociation. Even though the binding experiments showed that there are four sites for each substrate per mole of tetramer, the binding sites may not be intrinsic to each monomer383940414243. There are two possible reasons; one or more of the substrate sites may be formed as a result of conformational changes which are caused by the protein-protein interactions responsible for the association of monomers, or one or more of the substrate sites may possibly be located at the site of interaction between subunits and that the substrate site was composed of segments of polypeptide chains from two distinct subunits. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: The objective was to design a biocatalyst for myltet rahydrofolate ligase from the sequ- ences of archaea bacteria based on the evolutionar y conser vation at metal- and subst rate-binding regions. Methods: Complete for myltet rahydrofolate ligase sequences of archaea were ret rieved from Gen- Pept of National Center for Biotechnology Infor mation ( NCBI). The best st r uct ure identit y and the shor test metal- and subst rate-binding regions with templates were concer ned for generating homology models and then selected for docking with respective subst rates using AutoDock. Ever y homology model of this st udy was energy minimized to generate the lowest energetic confor mers. The best enzyme-subst rate complex models were chosen based on the mode of catalysis, t ypes of molecular interactions, and binding aff init y. Results: Two stable biocatalyst variants were developed from the sequences of Haloquadratum walsbyi DSM 16790 and Methanocorpusculum labreanum Z. The binding energy of variant 1-subst rate complex and variant 2-subst rate complex were ranged from -5.74 to -3.27 kcal /mol and from -5.23 to -2.78 kcal /mol, respectively. The most impor tant interactions cont ributing to the high binding affinity occur bet ween 5, 10-methenyltet rahydrofolate and the side chains of Glu113, Ser115 and Asp147 in variant 1 and Glu100, Ser102 and Asp134 in variant 2. Both variants have cesium- and subst rate-binding sites within 30 amino acids length wherein enzyme active sites residues were noted and thus evolutionar y conser vation in sequence as well as st r uct ure would make contribution in enzyme catalysis. Conclusion: This computer-aided protein designing approach is merely based on evolutionar y hypothesis. It is also const r uctive to develop a stable biocatalyst variant with more catalytic eff iciency because original conser ved amino acids residues are preser ved as it is. Both variants have catalytic f unctions as similar to 5-hydrolyase, ammonia for ming and amino lyase on 5, 10-methenyltet rahydrofolate.
    Full-text · Article · Jun 2011 · Functional & Integrative Genomics
    • "2b) correspond in 5-FCLs to the core of the active site that binds both 5-CHO-THF and ATP (Chen et al. 2004; Chen et al. 2005). In 5-FCLs, the penultimate residue of this conserved set is tyrosine, and changing it to alanine causes almost total (97–99%) loss of 5-FCL activity (Field et al. 2007; Wu et al. 2009). In contrast, the penultimate residue in COG0212 proteins is typically alanine or serine and never tyrosine (Fig. 2b). "
    [Show abstract] [Hide abstract] ABSTRACT: A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.
    Full-text · Article · May 2011
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