title: Cloning, purification and preliminary crystallographic analysis of Ara127N, a GH127 β-L-arabinofuranosidase from Geobacillus stearothermophilus T6.
abstract: The L-arabinan utilization system of Geobacillus stearothermophilus T6 is composed of five transcriptional units that are clustered within a 38 kb DNA segment. One of the transcriptional units contains 11 genes, the last gene of which (araN) encodes a protein, Ara127N, that belongs to the newly established GH127 family. Ara127N shares 44% sequence identity with the recently characterized HypBA1 protein from Bifidobacterium longum and thus is likely to function similarly as a β-L-arabinofuranosidase. β-L-Arabinofuranosidases are enzymes that hydrolyze β-L-arabinofuranoside linkages, the less common form of such linkages, a unique enzymatic activity that has been identified only recently. The interest in the structure and mode of action of Ara127N therefore stems from its special catalytic activity as well as its membership of the new GH127 family, the structure and mechanism of which are only starting to be resolved. Ara127N has recently been cloned, overexpressed, purified and crystallized. Two suitable crystal forms have been obtained: one (CTP form) belongs to the monoclinic space group P21, with unit-cell parameters a = 104.0, b = 131.2, c = 107.6 Å, β = 112.0°, and the other (RB form) belongs to the orthorhombic space group P212121, with unit-cell parameters a = 65.5, b = 118.1, c = 175.0 Å. A complete X-ray diffraction data set has been collected to 2.3 Å resolution from flash-cooled crystals of the wild-type enzyme (RB form) at -173°C using synchrotron radiation. A selenomethionine derivative of Ara127N has also been prepared and crystallized for multi-wavelength anomalous diffraction (MAD) experiments. Crystals of selenomethionine Ara127N appeared to be isomorphous to those of the wild type (CTP form) and enabled the measurement of a three-wavelength MAD diffraction data set at the selenium absorption edge. These data are currently being used for detailed three-dimensional structure determination of the Ara127N protein.
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: Roie
authors.initials: R
authors.lastname: Dann
authors.affiliation: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Izhak
authors.initials: I
authors.lastname: Shner
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 38000 Grenoble, France.
authors.forename: Babu A
authors.initials: BA
authors.lastname: Manjasetty
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 38000 Grenoble, France.
authors.forename: Hassan
authors.initials: H
authors.lastname: Belrhali
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: D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D017931:DNA Primers; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: Geobacillus stearothermophilus; MAD; SAD; arabinan utilization; arabinofuranoside; arabinose; glycoside hydrolase; selenomethionine; β-arabinofuranosidase, GH127
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