Site-Directed Mutagenesis of a Novel Serine Arylesterase fromVibrio mimicusIdentifies Residues Essential for Catalysis

Chang, Rey-Chang; Chen, Jack Chien; Shaw, Jei‐Fu

doi:10.1006/bbrc.1996.0620

Cited by 15 publications

(11 citation statements)

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“…Indeed, the equivalent of Gly-78 in other SGNH esterases has been implicated in allowing the nucleophilic Ser to adopt an unstrained conformation (8,28). Also, residues equivalent to Asp-79 have been proposed to participate in substrate binding in addition to appropriately positioning the Ser nucleophile (8,28,30). Our preliminary PG binding data support this additional role for Asp-79.…”

Section: Discussionsupporting

confidence: 58%

Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase

Pfeffer

Weadge²,

Clarke

2013

Journal of Biological Chemistry

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Background: O-Acetylpeptidoglycan esterase functions to release O-acetyl groups from peptidoglycan, thereby permitting the continued metabolism of this essential cell wall component. Results: Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism. Conclusion: Ape1a functions as a classical serine esterase. Significance: Ape represents a novel target for the development of new antibiotics.

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Section: Discussionsupporting

confidence: 58%

Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase

Pfeffer

Weadge²,

Clarke

2013

Journal of Biological Chemistry

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“…The deduced protein sequence of EstA does not exhibit the G-X-S-Y-G motif characteristically observed in eukaryotic and prokaryotic esterases and lipases (Cho and Cronan 1993;Jaeger et al 1994). Also, EstA does not have the G-D-S-L or G-D-S-L-S motifs which represent distinct subfamilies of serine-dependent esterases and lipases (Cho and Cronan 1993;Upton and Buckley 1995;Chang et al 1996;Carinato et al 1998). However, EstA does have a G-X-H motif located 22 residues from the COOH-terminus, which is characteristic of G-D-S-L and G-D-S-L-S type aryl-and thioesterases (Cho and Cronan 1993;Upton and Buckley 1995).…”

Section: Discussionmentioning

confidence: 94%

Characterization of an arylesterase from Lactobacillus helveticus CNRZ32

2000

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An esterase gene (estA) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The estA gene consisted of a 558 bp open reading frame encoding a putative peptide of 21·3 kDa. Protein sequence homology searches using BLAST revealed that EstA had low amino acid sequence identity with the serine‐dependent arylesterases TesI (24%) and EtpA (26%) from Escherichia coli and Vibrio mimicus, respectively. A recombinant EstA fusion protein containing a C‐terminal six‐histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstA revealed that it was a serine‐dependent enzyme having a monomeric Mr of 22·6–25·1 kDa. Optimum temperature, NaCl concentration and pH for EstA activity were determined to be 35–40 °C, 3·5% NaCl and 7·5–8·0, respectively. EstA had significant activity under conditions simulating those of ripening cheese (10 °C, 4% NaCl, pH 5·1). EstA hydrolysed a variety of ester compounds and preferred those with substituted phenyl alcohol and short‐chain fatty acid groups. Site‐directed mutagenesis suggested that the S10 and H164 residues were essential for EstA activity.

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“…The arylesterase from Acinetobacter sp. strain ADP1 (24) or V. mimicus (14) is a serine esterase containing a typical Ser-Asp-His catalytic triad in its active site as well. Judging from the alignment of the S. solfataricus P1 arylesterase amino acid sequence with those from Acinetobacter sp.…”

Section: Discussionmentioning

confidence: 99%

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

2008

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A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C 4 ) to palmitate (C 16 ), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k cat /K m ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s ؊1 ⅐ M ؊1 , respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.Sulfolobus solfataricus P1 (DSM1616), an extreme thermoacidophilic archaeon, inhabits sulfur-rich volcanic hot springs in a high-temperature and low-pH environment (10). Enzymes from thermoacidophilic archaea have attracted considerable attention among researchers because of the significance of their evolutionary phylogeny, the structural and functional stability of their enzymes despite their extreme environments, and the broad applications in biotechnology and industry available due to their special properties (23,44).Esterases (ester hydrolases) (EC 3.1

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Site-Directed Mutagenesis of a Novel Serine Arylesterase fromVibrio mimicusIdentifies Residues Essential for Catalysis

Cited by 15 publications

References 0 publications

Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase

Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase

Characterization of an arylesterase from Lactobacillus helveticus CNRZ32

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

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