Abstract
The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a alpha/beta hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 A resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a beta-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co2+. Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" esterases involved in plant cell wall degradation are shown to be closely related to the de-N-acetylases involved in chitin and peptidoglycan degradation (Blair, D. E., Schuettelkopf, A. W., MacRae, J. I., and Aalten, D. M. (2005) Proc. Natl. Acad. Sci. U. S. A., 102, 15429-15434), which form the NodB deacetylase "superfamily."
Highlights
The S. lividans enzyme is almost indistinct from known chitin/peptidoglycan deacetylases and is as efficient on those substrates as on xylan itself; the enzyme is, most likely to function in xylan degradation in vivo
Annotation of this enzyme as an acetylxylan esterase, as opposed to chitin deacetylase, was originally based upon its chromosomal organization and upon its enhanced expression when the organism is cultured on birchwood xylan (41)
Further evidence for a role in xylan degradation comes from the observation that the enzyme is appended to a CBM2 domain whose function could be the binding of crystalline substrates, the sequence of the CBM is most characteristic of a CBM2b xylan binding domain
Summary
Interaction, is fully consistent with both the specificity of this family of esterases for sugar-derived (as opposed to “generic”) esters, and the observation, in the case of SlCE4 (40), that catalysis demands a free vicinal OH group adjacent to the ester. A description of the metal ion geometry or any changes in such coordination during catalysis is difficult. The streptococcal enzyme displays octahedral metal ion coordination in its acetate complex, yet it is tetrahedral in complex with sulfate (19), suggesting that changes in geometry may occur during catalysis.
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