|Ph.D Student||Alalouf Onit|
|Subject||Biochemical Characterization and Structure-Function Studies|
of Acetylxylan Esterases from
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
The plant cell wall is composed of three major polysaccharides: cellulose, hemicellulose, and pectin. Esterification of the carbohydrates moieties by acetic acid prevents their hydrolysis by invading microorganisms, and contributes to polysaccharide hydration and chain separation. Microorganisms use acetylxylan esterases to remove acetyl groups from position 2 and/or 3 of the xylopyranosyl residues of the hemicellulose xylan to facilitate its hydrolysis by xylanases. Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic bacterium that degrades xylan using an extensive hemicellulolytic system. Two genes, axe1 and axe2, in the hemicellulolytic system encode putative acetyl-xylan esterases. Based on their amino acid sequences Axe1 and Axe2 are intracellular proteins belonging to carbohydrate esterase family 4 (CE4) and the SGNH hydrolases superfamily, respectively. In this study Axe1 and Axe2 were characterized biochemically and structurally.
Both axe1 and axe2 genes are induced by xylose and the gene products are O-deacetylases. Axe1 hydrolyzed 65% of the acetyl groups on fully acetylated xylan, and removed acetyl groups from partially acetylated 3,4-di-O-Acetyl-met-β-D-xylopyranoside, and mono-acetylated sugar(s). Axe2 was not active on acetylated xylan but was capable of completely removing acetyl groups from fully acetylated xylobiose, methyl-β-D-glucopyranoside, and methyl-β-D-xylopyranoside, the latter occurs through two intermediates: 3,4-di-O-acetyl-methyl-β-D-xylopyranoside and 2-mono-O-acetyl-methyl-β-D-xylopyranoside. Overall, Axe1 deacetylates larger oligomers than Axe2 explaining the need for two intracellular acetylxylan esterases by the organism.
The serine esterase mechanism of Axe2 was confirmed by site directed mutagenesis experiments and crystal structure analysis. Replacing each of the three putative catalytic residues, S15A, D191A, or H194A, abolished the catalytic activity of the enzyme. Residues Ser-15, Gly-63, and Asn-92 construct the oxyanion hole and Asp-191 play a role in maintaining His-194 in the correct position for catalysis. The catalytic constants of Axe2 on 2-naphthyl acetate demonstrated that the affinity of the enzyme toward the substrate is strongly affected by the pH, the rate limiting step is probably the second deacetylation step, and that the enzyme is not sensitive towards specific esterases/proteases inhibitors.
The crystal structures of Axe2 show a α/β/α fold consisting of five central parallel β-sheets, flanked by two layers of helices. Axe2 is a doughnut-like shaped octamer, composed of two staggered tetrameric rings stabilized mainly by clusters of hydrogen bonds (Tyr-184 of one subunit and Arg-192 and Asp-145 of another subunit). A single replacement, Y184F or R192K, converted the octameric structure into a dimer. The activity of the Axe2 dimer was much lower than that of the WT suggesting that the octameric arrangement is stabilizing the catalytic loop.