RESUMO
Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.
Assuntos
Hordeum/metabolismo , Proteínas de Plantas , Estrutura Terciária de Proteína , Serina Endopeptidases , Subtilisina/antagonistas & inibidores , Inibidor da Tripsina de Soja de Kunitz , alfa-Amilases/antagonistas & inibidores , Cristalografia por Raios X , Análise Mutacional de DNA , Detergentes/química , Endopeptidase K/antagonistas & inibidores , Metais/química , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Dobramento de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Inibidor da Tripsina de Soja de Kunitz/química , Inibidor da Tripsina de Soja de Kunitz/genética , Inibidor da Tripsina de Soja de Kunitz/metabolismoRESUMO
An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mgL(-1) from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mgL(-1), respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to possess the same characteristics as wild-type protein purified from barley grains.
