title: Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library.
abstract: A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K m and k cat of 39 μM and 25.8 s(-1), respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80-114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.
authors: Norwegian College of Fishery Science, University of Tromsø, 9037 Tromsø, Norway. juan.fu@uit.no
authors: Juan
authors: J
authors: Fu
authors: Hanna-Kirsti S
authors: HK
authors: Leiros
authors: Donatella
authors: D
authors: de Pascale
authors: Kenneth A
authors: KA
authors: Johnson
authors: Hans-Matti
authors: HM
authors: Blencke
authors: Bjarne
authors: B
authors: Landfald
mesh_terms: D001110:Arctic Regions; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism*; D015723:Gene Library; D008049:Lipase / Q000737:chemistry / Q000378:metabolism; D056186:Metagenomics / Q000379:methods*; D010802:Phylogeny; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology
chemical_list: D004950:Esterases; D008049:Lipase