Probing Enzyme Promiscuity of SGNH Hydrolases

• Published in: Chembiochem : a European journal of chemical biology Vol. 11; no. 15; pp. 2158 - 2167
• Main Authors: Leščić Ašler, Ivana, Ivić, Nives, Kovačić, Filip, Schell, Sabrina, Knorr, Janina, Krauss, Ulrich, Wilhelm, Susanne, Kojić-Prodić, Biserka, Jaeger, Karl-Erich
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 18.10.2010; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Amino Acid Sequence; autotransporter proteins; Bacterial Proteins - chemistry; Bacterial Proteins - classification; Bacterial Proteins - genetics; bacterial SNGH hydrolases; Butyrates - chemistry; Butyrates - metabolism; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; enzyme catalysis; Hydrolases - chemistry; Hydrolases - classification; Hydrolases - metabolism; Kinetics; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Molecular Sequence Data; Palmitoyl-CoA Hydrolase - chemistry; Palmitoyl-CoA Hydrolase - genetics; Palmitoyl-CoA Hydrolase - metabolism; Phylogeny; promiscuity; Protein Structure, Tertiary; selectivity; Sequence Alignment; Substrate Specificity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201000398

Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstAN-EstPC and EstPN-EstAC were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.