Active-site characterization of Candida boidinii formate dehydrogenase

2001 ◽  
Vol 354 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Nikolaos E. LABROU ◽  
Daniel J. RIGDEN
Keyword(s):  
Nucleotide Sequence ◽  
Molecular Modelling ◽  
Formate Dehydrogenase ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Candida Boidinii ◽  
Three Dimensional Model ◽  
Embl Nucleotide Sequence Database ◽  
Anchor Group

NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii was cloned and expressed to a high level in Escherichia coli (20% of soluble E. coli protein). Molecular modelling studies were used to create a three-dimensional model of C. boidinii FDH, based on a known structure of the Pseudomonas sp. 101 enzyme. This model was used for investigating the catalytic mechanism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were characterized by steady-state kinetic analysis: the wild type as well as 10 mutants involving single (Phe-69–Ala, Asn-119–His, Ile-175–Ala, Gln-197–Leu, Arg-258–Ala, Gln-287–Glu and His-311–Gln) and double amino acid substitutions (Asn-119–His/His-311–Gln, Gln-287–Glu/His-311–Gln and Gln-287–Glu/Pro-288–Thr). The kinetic results of the mutant enzymes provide the first experimental support that hydrophobic patches, formed by Phe-69 and Ile-175, destabilize substrates and stabilize products. Also, the key role of Arg-258 in stabilization of the negative charge on the migrating hydride was established. Asn-119, besides being an anchor group for formate, also may comprise one of the hinge regions around which the two domains shift on binding of NAD+. The more unexpected results, obtained for the His-311–Gln and Gln-287–Glu/His-311–Gln mutants, combined with molecular modelling, suggest that steric as well as electrostatic properties of His-311 are important for enzyme function. An important structural role has also been attributed to cis-Pro-288. This residue may provide the key residues Gln-287 and His-311 with the proper orientation for productive binding of formate. The FDH nucleotide sequence has been submitted to the EMBL Nucleotide Sequence Database under the accession no. AJ011046.

Mechanistic and inhibition studies of chorismate-utilizing enzymes

2005 ◽  
Vol 33 (4) ◽  
pp. 763-766 ◽  
Author(s):  
O. Kerbarh ◽  
E.M.M. Bulloch ◽  
R.J. Payne ◽  
T. Sahr ◽  
F. Rébeillé ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Higher Plants ◽  
Three Dimensional ◽  
Antimicrobial Effect ◽  
Three Dimensional Model ◽  
Antimicrobial Drugs ◽  
Apicomplexan Parasites ◽  
Covalent Intermediate ◽  
Inhibition Studies ◽  
First Time

The shikimate biosynthetic pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites; it involves seven enzymatic steps in which phosphoenolpyruvate and erythrose 4-phosphate are converted into chorismate. In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin. As mammals do not possess such a biosynthetic system, the enzymes involved in the pathway have aroused considerable interest as potential targets for the development of antimicrobial drugs and herbicides. As an initiative to investigate the mechanism of some of these enzymes, we showed that the antimicrobial effect of (6S)-6-fluoroshikimate is the result of irreversible inhibition of 4-amino-4-deoxychorismate synthase by 2-fluorochorismate. Based on this study, a catalytic mechanism for this enzyme was proposed, in which the residue Lys-274 is involved in the formation of a covalent intermediate. In another study, Yersinia enterocolitica Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin, was for the first time biochemically characterized and shown to catalyse the formation of salicylate from chorismate via isochorismate as a reaction intermediate. A three-dimensional model for this enzyme was constructed that will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake.

scholarly journals Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin β-Ketoacyl-Acyl Carrier Protein Synthase

2000 ◽  
Vol 182 (9) ◽  
pp. 2619-2623 ◽  
Author(s):  
Min He ◽  
Mustafa Varoglu ◽  
David H. Sherman
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Three Dimensional Model ◽  
Catalytic Active Site ◽  
Ketoacyl Synthase

ABSTRACT A three-dimensional model of the Streptomyces coelicolor actinorhodin β-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the relatedEscherichia coli fatty acid synthase condensing enzyme β-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. 3H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

2006 ◽  
Vol 387 (5) ◽  
pp. 515-523 ◽  
Author(s):  
Shivakumara Bheemanaik ◽  
Janusz M. Bujnicki ◽  
Valakunja Nagaraja ◽  
Desirazu N. Rao
Keyword(s):  
Dna Methyltransferase ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Cofactor Binding ◽  
Protein Fold Recognition ◽  
Fluorescence Measurements ◽  
Complete Disruption

AbstractKpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5′-GGTACC-3′ and transfers the methyl group fromS-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation andin vitrorestriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.

scholarly journals Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-l-alanine amidase

2004 ◽  
Vol 377 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Catherine GÉNÉREUX ◽  
Dominique DEHARENG ◽  
Bart DEVREESE ◽  
Jozef VAN BEEUMEN ◽  
Jean-Marie FRÈRE ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Hydrolytic Enzymes ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Activity

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-l-alanine amidase involved in both peptidoglycan recycling and β-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+N-acetylmuramyl-l-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.

scholarly journals Basic residues play key roles in catalysis and NADP+-specificity in maize (Zea mays L.) photosynthetic NADP+-dependent malic enzyme

2004 ◽  
Vol 382 (3) ◽  
pp. 1025-1030 ◽  
Author(s):  
Enrique DETARSIO ◽  
Carlos S. ANDREO ◽  
María F. DRINCOVICH
Keyword(s):  
Zea Mays ◽  
Malic Enzyme ◽  
Zea Mays L ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Reciprocal Inhibition ◽  
Partition Ratio ◽  
Site Directed Mutagenesis ◽  
Three Dimensional Model ◽  
Coenzyme Specificity

C4-specific (photosynthetic) NADP+-dependent malic enzyme (NADP+-ME) has evolved from C3-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP+-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP+-ME K255I (Lys-255→Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP+-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play ‘back-up’ roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP+ versus NAD+) of maize NADP+-ME by interacting with the 2′-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP+ or NAD+, as well as by the reciprocal inhibition constants of the competitive inhibitors 2′-AMP and 5′-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436→Ile) with wild-type NADP+-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP+-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.

scholarly journals A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

2004 ◽  
Vol 279 (44) ◽  
pp. 46172-46181 ◽  
Author(s):  
Stéphanie Voisin ◽  
Didier Rognan ◽  
Claude Gros ◽  
Tanja Ouimet
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Physiological Role ◽  
Site Directed Mutagenesis ◽  
Three Dimensional Model ◽  
X Ray ◽  
Amide Bonds ◽  
Inhibitory Compounds ◽  
Proposed Model

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of thein vitroenzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S′2subsite of NEP2 where Ser133and Leu739replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg131and Asn567in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S′2pocket, particularly the presence therein of Leu739, account for a number of differences in inhibitor binding between NEP and NEP2.

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