Crystal structure of engineered β‐glucosidase from a soil metagenome

摘要

Intensive screening of microbial isolates over the last several years has resulted in the identification and commercialization of numerous biomolecules, many of which are the products of microbial secondary metabolism.1 Soil microorganisms in particular have been identified as valuable sources of naturally occurring industrially relevant antibiotics.2 It has been estimated, based on the reassociation kinetics of DNA isolated from various soil samples, that the number of distinct prokaryotic genomes in soil ranges from 2000 to 18,000 genomes per gram of soil.3 Metagenomics is a rapidly developing field that involves the study of complex genomes within different environments and microbial niches.4 Several successful attempts to generate novel enzymes with enhanced catalytic activity and thermal stabilities using metagenomics have recently been reported.5-7 The utilization of polysaccharides as an energy, chemical, and carbon source requires the activity of β-glucosidase (Bgl; EC 3.2.1.21).8, 9 Bgl cleaves β-1,4-glycosidic linkages in disaccharide or glucose-substituted molecules through an acid–base catalytic mechanism, analogous to hydrolysis.10 The function of Bgl is essential for carbohydrate metabolism in cells, and the use of Bgl in various biotechnological processes, such as food and chemical synthesis, has been explored.11, 12 Defects in Bgl activity in humans are associated with Gaucher disease, a non-neuronopathic lysosomal storage disorder.13 There are two Bgl homologues, BglA and BglB, which have been shown to be members of the GH-1 family of enzymes. BglA and BglB are involved in the hydrolysis of cellodextrins, but they have different quaternary structures and substrate specificities. BglA is a cellobiase with an unusual octameric structure, while BglB is a monomer that acts as an exo-Bgl, hydrolyzing cellobiose and cellodextrins with a high degree of polymerization.14, 15 Recently, we isolated and characterized a novel bgl gene from uncultured soil bacteria (Usbgl).16 The gene encoded a protein with a predicted molecular weight of 55 kDa and an amino acid sequence was 56% identical to the family 1-glycosyl hydrolase of Chloroflexus aurantiacus. UsBgl exhibited substantial glycosyl hydrolase activity in the presence of natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, salicin, and arbutin, and was able to convert the major ginsenoside Rb1 into the pharmaceutically active minor ginsenoside, Rd. UsBgl also exhibited thermostable properties, which indicated that it may be useful in the development of novel biotechnical processes.16 Here, we report the X-ray crystal structure of engineered UsBgl. This type of structural analysis will contribute not only to our ability to engineer pharmaceutically active forms Bgl, but may also lead to a better understanding of the molecular etiology of Gaucher disease. Our results also provide insight into the development and production of value-added products, including pharmaceuticals. Recombinant Bgl that lacked a signal peptide (accession no. DQ842022) was amplified from the β-glucosidase gene using the primers 5′-TCGCGGATCCATGACTGAACATGAGCTTCAG-3′ (which contained a BamHI site at the 5′-end) and 5′GCAAGCTTAATAGCGGGCG CGGCTAGCCC-3′ (which contained a HindIII site at the 3′-end). The amplified DNA was ligated into BamHI- and HindIII -digested pET28a(+) (Novagen) to create pEGLU. Mutations were introduced using the QuikChange Multi Site-Directed Mutagenesis kit (Stratagene) and pEGLU (encoding wild-type UsBgl) as the template. Two primers were designed to introduce the following sets of mutations: N-terminal primer (5′-TC GGA TCC AAC GTT AAG AAG TTC CCC GAG GGC TTT CTG TGG GGC-3′) introduced the mutations Glu39AsnN, Leu40Val, Gln41Lys, Pro42Lys, and Lys45Glu; C-terminal primer (5′-GC AAG CTT TCA ATC CTC TAG CCC GTT GTT CGC AAT TAC GTC GCG-3′) introduced the mutations Arg477Asn, Ala481Glu, and Ala482Asp. BL21(DE3) cells were transformed using recom