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 Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

1999 ◽  
Vol 43 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Sonia Trépanier ◽  
James R. Knox ◽  
Natalie Clairoux ◽  
François Sanschagrin ◽  
Roger C. Levesque ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
Acid Group ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Class C ◽  
Three Dimensional Models ◽  
Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

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 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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