engleski

How promiscuous are SGNH-hydrolases?

Although lipolytic enzymes are known for decades, the classification of this type of enzymes is a continuous process. Among many defined classes and families, a new one was recently identified: SGNH-hydrolases (1). According to InterPro database (March 2008.), there are 3775 members of this family, with protein sequences originating mainly from genome sequencing projects. However, only 10 SGNH-hydrolases are crystallized and their three-dimensional structures solved (coordinates in PDB database, March 2008.), and very few are biochemically characterized to some extent. The most thoroughly characterized member of this family is thioesterase I/protease I/lysophospholipase L1 from Escherichia coli. Data on substrate specificity of this enzyme show its pronounced substrate promiscuity (2). To examine possible multifunctionality of SGNH-hydrolases, we performed substrate specificity analysis on several members of this family: lipase from Streptomyces rimosus (SrLip), esterases from Pseudomonas aeruginosa and Pseudomonas putida (EstA and EstP, respectively), and acyl-CoA thioesterase from P. aeruginosa (TesA). Several types of substrates were tested (34 in total): for esterase, thioesterase, lipase, phospholipase, Tweenase and protease activity. Among tested enzymes, only SrLip showed significant activity towards a broad spectrum of substrates. TesA was most active towards Tween-detergents and short-chained esters, and EstA and EstP showed activity profiles typical for esterases. After measuring enzyme activity at different substrate concentrations, it was observed that investigated enzymes, characterized as esterases, show classical Michaelis-Menten kinetics, only SrLip showed pronounced interfacial activation. Our findings show that the lipase from Streptomyces rimosus does reveal multifunctionality, i.e. activity towards several substrate types, especially high towards phospholipase substrates and triglycerides. References [1] Molgaard, A., Kauppinen, S., Larsen, S. (2000): Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Structure 8: 373-383. [2] Karasawa, K., Yokoyama, K., Setaka, M., Nojima, S. (1999): The Escherichia coli pldC gene encoding lysophospholipase L1 is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively. J. Biochem. 126: 445-448.

bacterial SGNH-hydrolases; multifunctionality; enzyme kinetics

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