Roles of Dimerization Domain Residues in Binding and Catalysis by Aminoacylase-1

Lindner, Holger A.; Alary, Alain; Boju, Lorena; Sulea, Traian; Ménard, Robert

doi:10.1021/bi051180y

Cited by 25 publications

(38 citation statements)

References 30 publications

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“…2). Based on the loss of activity of the D284A mutant and on the similar results obtained for hAcy1 (35), it can be deduced that Asp 284 plays an essential role in correct positioning of Arg 286 for substrate binding. We suspect that the partial loss of activity of the T261A mutant was due to partial disruption of the binding network with Arg 234 ; mutation of this Thr was not sufficient to produce changes in BsLcar oligomerization, but it did alter the position of the loop containing His 225 , as Arg 234 also seems to anchor/position this structural element (loop containing ␤8 in Fig.…”

Section: R369 H219(a) N260(a)supporting

confidence: 62%

“…In fact, the Arg and His residues are strictly conserved. Based on the literature available for homologous enzymes, the conserved Arg residue is or might be necessary for substrate binding in ␤-alanine synthase (4,37,38), EcAam (2), peptidase V (PepV) (31), carnosinase (55), IAA-amino acid hydrolase homolog 2 (ILL2) (7), peptidase D (PepD) (9), DapE (46), hAcy1 (35), peptidase T (PepT) (6,24), and metallopeptidase (Sapep) (20). Several homologous structures present different molecules bound to these residues, namely, the structures under PDB IDs 1VIX, 1FNO, 3IC1, 3IFE, and 2QYV.…”

Section: R369 H219(a) N260(a)mentioning

confidence: 99%

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Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

et al. 2012

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c N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg 234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family. N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases; EC 3.5.1.87) irreversibly hydrolyze the amide bond of the carbamoyl group in L-N-carbamoyl-amino acids. Due to their stereospecificity, L-carbamoylases are widely used in kinetic resolution for the production of optically pure amino acids (see reference 44 and references therein). Furthermore, their substrate promiscuity allows their use in different multienzymatic processes, increasing their economic and industrial relevance (48). L-Carbamoylases have been isolated and characterized from different sources, although most of the studies carried out have focused on biotechnological applications (44). Their relationship with other amidohydrolases and with the peptidase M20/M25/M40 family has been established based on sequence similarity (21,29,42). A closer relationship among L-carbamoylases and ␤-ureidopropionases is supported by the findings of two ␤-ureidopropionases that are able to hydrolyze N-carbamoyl-L-␣-amino acids (40,41,47).In a previous work, we were able to show the involvement in catalysis of several highly conserved residues in L-carbamoylases, as well as the relationship of these enzymes with several peptidases (42). Dimerization was also proved to be necessary for enzymatic activity, as the residues involved in catalysis come from both monomers. In this work, we determined the first crystal structure of an L-carbamoylase, belonging to Geobacillus stearothermophilus CECT43 (BsLcar). Mutagenesis studies of BsLcar confirmed the importance of conserved residues in this enzyme. Unexpectedly, a highly conserved arginine in L-carbamoylases has been proved critical for the dimerization of the enzyme, and thus for enzymatic activity. Comparative studies revealed striking characteristics of the different "dimerization" domains of the peptidase M20/M25/ M40 family, in agreement with previous evolutionary hypotheses on this family of enzymes (3, 37) and suggesting new evolutionary considerations. Finally, an alternative re...

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Section: R369 H219(a) N260(a)supporting

confidence: 62%

Section: R369 H219(a) N260(a)mentioning

confidence: 99%

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

et al. 2012

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“…In addition to determining the enzymatic activity of the purified AAIII dimer, it is also necessary to determine whether a single AAIII monomer possesses the enzymatic activity. It should be mentioned that AAI, known to form a homodimer (Kordel and Schneider, 1976;D'Ambrosio et al, 2003;Lindner et al, 2003), also demonstrates Michaelis kinetics (Uttamsingh et al, 1998;Lindner et al, 2005).…”

Section: Specificity Of Murine Aminoacylase IIImentioning

confidence: 99%

“…It contains one Zn 2ϩ per monomer of molecular mass ϳ43 kDa. AAI isolated from different sources was shown to be a homodimer (Kordel and Schneider, 1977;Lindner et al, 2003Lindner et al, , 2005. AAI was shown to deacetylate several…”

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confidence: 99%

Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

et al. 2006

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ABSTRACT:Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-L-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate ␤-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V max value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of ␤-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto-and nephrotoxicity.Trichloroethylene (TCE) and other industrial solvents are shown to cause nephrotoxicity and hepatotoxicity, and are probable human carcinogens (Maltoni et al., 1988;Cummings and Lash, 2000). One of the TCE toxicity mechanisms involves the conjugation with glutathione, which is transformed by ␥-glutamyltransferase and dipeptidase to cysteine S-conjugates Berrnauer et al., 1996;Anders and Dekant, 1998). The formation of cysteine S-conjugates takes place mainly in liver and, to some extent, also in kidney. Cysteine S-conjugates may be acetylated in the liver and kidney into corresponding N-acetyl S-conjugates (mercapturic acids) and transported from the liver into the kidney. In the kidney proximal tubules, mercapturic acids may be secreted or deacetylated by aminoacylases into cysteine S-conjugates Dekant, 1994, 1998). The deacetylation reaction may play an important role in the renal nephrotoxicity since cysteine S-conjugates, but not mercapturates, are transformed by cysteine conjugate ␤-lyases into toxic metabolites (Hayden and Stevens, 1990;Commandeur et al., 1995). Cysteine S-conjugates also may be transformed by flavoprotein monooxygenase...

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“…The enzyme is a homodimer with a molecular mass of 90 kDa [15] and follows a zinc-based, general base-like catalytic mechanism [23]. As a special feature of the M20 family of homodimeric enzymes, the active site in pAcy1 is situated at the subunit interface [23,24]. N-acyl-L-amino acid binding to the enzyme places the scissile bond of the substrate in the vicinity of a zinc-bound water molecule.…”

Section: Introductionmentioning

confidence: 99%

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis

et al. 2010

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Roles of Dimerization Domain Residues in Binding and Catalysis by Aminoacylase-1

Cited by 25 publications

References 30 publications

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis

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