Montoro-Garcia S

References (3)

Title : The crystal structure of the cephalosporin deacetylating enzyme acetyl xylan esterase bound to paraoxon explains the low sensitivity of this serine hydrolase to organophosphate inactivation - Montoro-Garcia_2011_Biochem.J_436_321
Author(s) : Montoro-Garcia S , Gil-Ortiz F , Garcia-Carmona F , Polo LM , Rubio V , Sanchez-Ferrer A
Ref : Biochemical Journal , 436 :321 , 2011
Abstract : Organophosphorus insecticides and nerve agents irreversibly inhibit serine hydrolase superfamily enzymes. One enzyme of this superfamily, the industrially important (for beta-lactam antibiotic synthesis) AXE/CAH (acetyl xylan esterase/cephalosporin acetyl hydrolase) from the biotechnologically valuable organism Bacillus pumilus, exhibits low sensitivity to the organophosphate paraoxon (diethyl-p-nitrophenyl phosphate, also called paraoxon-ethyl), reflected in a high K(i) for it (~5 mM) and in a slow formation (t((1/2))~1 min) of the covalent adduct of the enzyme and for DEP (E-DEP, enzyme-diethyl phosphate, i.e. enzyme-paraoxon). The crystal structure of the E-DEP complex determined at 2.7 A resolution (1 A=0.1 nm) reveals strain in the active Ser(1)(8)(1)-bound organophosphate as a likely cause for the limited paraoxon sensitivity. The strain results from active-site-size limitation imposed by bulky conserved aromatic residues that may exclude as substrates esters having acyl groups larger than acetate. Interestingly, in the doughnut-like homohexamer of the enzyme, the six active sites are confined within a central chamber formed between two 60 degrees -staggered trimers. The exclusive access to this chamber through a hole around the three-fold axis possibly limits the size of the xylan natural substrates. The enzyme provides a rigid scaffold for catalysis, as reflected in the lack of movement associated with paraoxon adduct formation, as revealed by comparing this adduct structure with that also determined in the present study at 1.9 A resolution for the paraoxon-free enzyme.
ESTHER : Montoro-Garcia_2011_Biochem.J_436_321
PubMedSearch : Montoro-Garcia_2011_Biochem.J_436_321
PubMedID: 21382014
Gene_locus related to this paper: bacpu-AXE

Title : Characterization of a novel thermostable carboxylesterase from Geobacillus kaustophilus HTA426 shows the existence of a new carboxylesterase family - Montoro-Garcia_2009_J.Bacteriol_191_3076
Author(s) : Montoro-Garcia S , Martinez-Martinez I , Navarro-Fernandez J , Takami H , Garcia-Carmona F , Sanchez-Ferrer A
Ref : Journal of Bacteriology , 191 :3076 , 2009
Abstract : The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65 degrees C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new alpha/beta hydrolase family different from IV and VI.
ESTHER : Montoro-Garcia_2009_J.Bacteriol_191_3076
PubMedSearch : Montoro-Garcia_2009_J.Bacteriol_191_3076
PubMedID: 19304850
Gene_locus related to this paper: geotn-a4isp0

Title : A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate - Martinez-Martinez_2007_Anal.Biochem_369_210
Author(s) : Martinez-Martinez I , Montoro-Garcia S , Lozada-Ramirez JD , Sanchez-Ferrer A , Garcia-Carmona F
Ref : Analytical Biochemistry , 369 :210 , 2007
Abstract : A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.
ESTHER : Martinez-Martinez_2007_Anal.Biochem_369_210
PubMedSearch : Martinez-Martinez_2007_Anal.Biochem_369_210
PubMedID: 17651681