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Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively.

Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure-function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phospho-transferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-β-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-β-galactosidase/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chaincleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of -xylosidases. One of these -xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombic P2 1 2 1 2 1 space group, with average unit-cell parameters a = 97.7, b = 119.1, c = 242.3 Å . Several X-ray diffraction data sets have been collected from flashcooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

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