product of hydrolysis of pNP-esters Leaving group of paraoxon when inhibiting enzymes. Product of hydrolysis of Esters of 4-Nitrophenol. Amounts of 4-nitrophenol produced by a particular enzyme in the presence of its corresponding substrate can be measured with a spectrophotometer at or around 405 nm and used as a proxy measurement for the amount of the enzyme activity in the sample. Leaving group of NEMP, Methylparaoxon, VXS
8 structures(e.g. : 8EA1, 7ATF, 7NB5... more)(less)8EA1: Structure of kudzu 2-hydroxyisoflavanone dehydratase in complex with P-nitrophenol, 7ATF: Structure of EstD11 in complex with p-Nitrophenol, 7NB5: Structure of EstD11 S144A in complex with naproxen p-nitrophenol ester, 6KF1: Microbial Hormone-sensitive lipase E53 mutant S162A, 5XH2: Crystal structure of a novel PET hydrolase R103G/S131A mutant in complex with pNP from Ideonella sakaiensis 201-F6, 5HC2: Structure of uncultured bacteria esterase Est22 (S188A) with p-nitrophenol, 5HC0: Structure of uncultured bacteria esterase Est22 with p-nitrophenol, 3A70: Crystal structure of Pseudomonas sp. MIS38 lipase in complex with diethyl phosphate
A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the alpha/beta-hydrolase superfamily. The canonical alpha/beta-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11
Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 A to 2.43 A. The Est22 exhibits a alpha/beta-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases.
