Crystallization and preliminary X-ray crystallographic analysis of a novel α-L-arabinofuranosidase (CtGH43) fromClostridium thermocellumATCC 27405

Abstract

The truncated carbohydrate-active enzyme belonging to family 43 glycoside hydrolase from Clostridium thermocellum (CtGH43) is an -l-arabinofuranosidase that in combination with endoxylanase leads to complete breakdown of l-arabinosyl-substituted xylans. The recombinant enzyme CtGH43 from C. thermocellum was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. The recombinant CtGH43 has a molecular mass of 35.86 kDa. Preliminary structural characterization was carried out on CtGH43 crystallized from different conditions, which gave either cube-shaped or brick-shaped crystals. These diffracted to a resolution of 1.65 Å for the cubic form and 1.1 Å for the monoclinic form. Molecular replacement was used to solve the CtGH43 structure.

Molecular determinants of substrate specificity revealed by the structure ofClostridium thermocellumarabinofuranosidase 43A from glycosyl hydrolase family 43 subfamily 16

“…Escherichia coli BL21 (DE3) pLysS cells harbouring pAG43 were cultured in LB containing 35 mg ml À1 chloramphenicol and 50 mg ml À1 kanamycin at 37 C to midexponential phase (A 600 = 0.5), at which point isopropyl -d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the cultures were incubated for a further 16 h at 24 C. The cells were collected by centrifugation and the cell pellet was resuspended in 50 mM HEPES buffer pH 7.5 containing 1 M NaCl, 5 mM CaCl 2 , 10 mM imidazole. Recombinant CtAbf43A was purified by immobilized metalaffinity chromatography (IMAC) as described previously (Goyal et al, 2014). To achieve a high degree of protein purity, gel filtration was included as a further purification step.…”

“…Full details of the crystallization, data collection and structure determination are given in Goyal et al (2014). Two forms of crystals of CtAbf43A were obtained: a cube-shaped crystal in 0.1 M sodium acetate pH 4.5, 2 M ammonium sulfate and brick-shaped crystals in 0.1 M Tris pH 8.5, 20% PEG 2000 monomethyl ether, 0.2 M trimethylamine N-oxide.…”