Probing Enzyme Promiscuity of SGNH Hydrolases

Abstract

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 EstA(N)-EstP(C) and EstP(N)-EstA(C) 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.

Structural and Biochemical Characterization of the Salicylyl-acyltranferase SsfX3 from a Tetracycline Biosynthetic Pathway

“…These studies demonstrate that SsfX3 has somewhat relaxed substrate specificity for both the acyl donors and acyl acceptors. However, the substrate specificity observed for SsfX3, although somewhat tolerant to closely related substrates, seems to be much more stringent when compared with the extremely broad range of substrates accepted by GDSL hydrolases reported by Asler et al (24). According to the crystal structure of SsfX3, this increased substrate specificity is likely influenced significantly by the putative N-terminal binding module, as discussed previously.…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…Some of the single-domain enzymes of this group are secreted and display PLA and LPLA activities, though a few also have glycerophospholipid cholesterol acyl transferase (GCATase; EC 2.3.1.43) activity, which cleaves phospholipids and transfers the acyl chain onto sterols (88). Some acyl esterases present in the OM of several species of Gammaproteobacteria have an additional C-terminal ␤-barrel domain that functions as an autotransporter (89). Both single-and two-domain acyl hydrolases of the SGNH esterase family have a flexible substrate-binding pocket and are active on a wide range of substrates (89).…”

“…Some acyl esterases present in the OM of several species of Gammaproteobacteria have an additional C-terminal ␤-barrel domain that functions as an autotransporter (89). Both single-and two-domain acyl hydrolases of the SGNH esterase family have a flexible substrate-binding pocket and are active on a wide range of substrates (89). Acyl hydrolases from the SGNH esterase family have been shown to be virulence factors in Moraxella bovis, Vibrio harveyi, Salmonella enterica, and Yersinia enterocolitica, as described below.…”

Enzyme Promiscuity: Engine of Evolutionary Innovation

“…Thus, the observed substrate range can be quite broad. Substrate specificity profiles have been reported for numerous enzyme classes including phosphatases (19 -23), thioesterases (24 -26), peptide racemases (27), and lipases/phospholipases (28). The substrate structures can be surprisingly varied; for instance, single enzymes can utilize long and short-chain acyl-CoA substrates (24), apolar and anionic lipids (28), or various length phosphosugars and nucleotides (20).…”