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Structure–function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus

DOI: 10.1107/S1399004715018672 DOI Help
PMID: 26627651 PMID Help

Authors: Hodaya V. Solomon (The Hebrew University of Jerusalem) , Orly Tabachnikov (Israel Institute of Technology) , Shifra Lansky (The Hebrew University of Jerusalem) , Rachel Salama (Israel Institute of Technology) , Hadar Feinberg (The Hebrew University of Jerusalem) , Yuval Shoham (Israel Institute of Technology) , Gil Shoham (The Hebrew University of Jerusalem)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Biological Crystallography , VOL 71 (12) , PAGES 2433 - 2448

State: Published (Approved)
Published: December 2015

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 -galactosidase capable of hydrolyzing short -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 / domain, and the smallest all- 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.

Journal Keywords: Geobacillus Stearothermophilus; Galactan Utilization; Galactosaccharides; Galactose; -Galactosidase; Glycoside Hydrolase; Gh42; Homotrimer; Catalytic Mutant; Glycosynthase

Subject Areas: Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography

Other Facilities: ESRF