title: Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.
abstract: Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Hodaya V
authors.initials: HV
authors.lastname: Solomon
authors.affiliation: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Orly
authors.initials: O
authors.lastname: Tabachnikov
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Shifra
authors.initials: S
authors.lastname: Lansky
authors.affiliation: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Rachel
authors.initials: R
authors.lastname: Salama
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Hadar
authors.initials: H
authors.lastname: Feinberg
authors.affiliation: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Gil
authors.initials: G
authors.lastname: Shoham
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009598:Nitrophenylgalactosides / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D021122:Protein Subunits; D011994:Recombinant Proteins; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D001616:beta-Galactosidase; D005690:Galactose
keywords: GH42; Geobacillus stearothermophilus; catalytic mutant; galactan utilization; galactosaccharides; galactose; glycoside hydrolase; glycosynthase; homotrimer; β-galactosidase