From a metagenomic source to a high-resolution structure of a novel alkaline esterase.
Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/β hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.
National Laboratory of Biosciences (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo State, Brazil.|University of São Paulo (USP), São Paulo, São Paulo State, Brazil.|Department of Technology, São Paulo State University (UNESP), Jaboticabal, São Paulo State, Brazil.
Mariana Rangel
MR
Pereira
University of São Paulo (USP), São Paulo, São Paulo State, Brazil.|Department of Technology, São Paulo State University (UNESP), Jaboticabal, São Paulo State, Brazil.
Thaís Carvalho
TC
Maester
National Laboratory of Biosciences (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo State, Brazil.|Institute of Biology, University of Campinas, Campinas, São Paulo State, Brazil.
Gustavo Fernando
GF
Mercaldi
Department of Technology, São Paulo State University (UNESP), Jaboticabal, São Paulo State, Brazil.
Eliana Gertrudes
EG
de Macedo Lemos
Department of Biochemistry, University of Cambridge, Cambridge, UK.
Marko
M
Hyvönen
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil. abalan@usp.br.
Andrea
A
Balan
D002087:Butyrates / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008066:Lipolysis; D056186:Metagenomics; D059013:Microbial Consortia / Q000235:genetics*; D009596:Nitrophenols / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002087:Butyrates; D009596:Nitrophenols; D011994:Recombinant Proteins; C033592:4-nitrophenyl butyrate; C008642:4-nitrophenyl acetate; D004950:Esterases; D008049:Lipase
Est8; Esterase; Metagenomic; Structure; p-NP esters