Heterologous expression, purification, crystallization, X-ray analysis and phasing of the acetyl xylan esterase from Bacillus pumilus.
Bacillus pumilus PS213 acetyl xylan esterase (AXE) acts as an accessory enzyme in the plant cell wall hemicellulose biodegradation pathway. It belongs to the carbohydrate esterase family 7 and hydrolyses the ester linkages of the acetyl groups in position 2 and/or 3 of the xylose moieties of the acetylated xylan fragments from hardwood. The enzyme displays activity towards a broad range of acetylated compounds including the antibiotic cephalosporin-C. In this study we report the heterologous expression, purification, physicochemical characterization and crystallization of the recombinant B. pumilus AXE. Remarkable improvement of the crystal quality was achieved by setting up crystallization conditions, at first established using the hanging drop vapor diffusion method, in a micro-batch experiment. Rod-like diffraction quality crystals were obtained using 10% PEG 6000, 0.1 M MES pH 6.0 and a wide range of LiCl concentrations (0.2-1.0 M) as precipitant agent. Two different crystal forms, both belonging to space group P2(1), were characterized, diffracting X-rays to 2.5 and 1.9 angstrom resolution. Successful molecular replacement showed 12 molecules in the asymmetric unit of either crystal forms that are arranged as two doughnut-like hexamers, each one encompassing a local 32 symmetry. A catalytic inactive mutant Ser181Ala of B. pumilus AXE was also engineered, expressed, purified and crystallized for functional and structural studies.
Krastanova
Ivet
I
International School for Advanced Studies, Via Beirut 2/4, I-34014 Trieste, Italy. kivet@sissa.it
Guarnaccia
Corrado
C
Zahariev
Sotir
S
Degrassi
Giuliano
G
Lamba
Doriano
D
D000115:Acetylesterase / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D002473:Cell Wall / Q000378:metabolism; D002511:Cephalosporins / Q000494:pharmacology; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D008027:Light; D018021:Lithium Chloride / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014961:X-Ray Diffraction; D014990:Xylans / Q000737:chemistry; D014994:Xylose / Q000737:chemistry
D002241:Carbohydrates; D002511:Cephalosporins; D011994:Recombinant Proteins; D014990:Xylans; C025163:cephalosporin C; D012694:Serine; D014994:Xylose; D004950:Esterases; D000115:Acetylesterase; C081851:acetylxylan esterase; D018021:Lithium Chloride; D000409:Alanine