The vicinal hydroxyl group is prerequisite for metal activation of Clostridium thermocellum acetylxylan esterase.
Positional specificity of NodB-like domain of a multidomain xylanase U from Clostridium thermocellum (CtAxe) was investigated. Of three monoacetates of 4-nitrophenyl beta-d-xylopyranoside the acetylxylan esterase domain showed a clear preference for the 2-acetate. Moreover, the enzyme was significantly activated by Co(2+). Acetylated methyl beta-d-xylopyranosides were deacetylated slightly better at position 3 than at position 2, suggesting that the enzyme binds the substrate with the small methyl aglycone also in the opposite orientation. Nevertheless, both positions 2 and 3 of methyl beta-d-xylopyranoside were deacetylated much faster in the presence of the activating metal ion. In contrast, replacement of the hydroxyl group at either of these positions with fluorine or hydrogen, as well as acetylation of both positions, abolished the enzyme activity, regardless the absence or the presence of Co(2+). Thus, the presence of the free vicinal hydroxyl group seems to be a prerequisite not only for an efficient deacetylation of position 2 or 3, but also for the activation of the enzyme with cobalt ion. The demonstrated involvement of the vicinal hydroxyl groups in the mechanism of deacetylation is in accord with 3-D structures of CtAxe as well as other CE4 metal-dependent deacetylases.
Biely
Peter
P
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia.
Mastihubová
Mária
M
Puchart
Vladimír
V
D000107:Acetylation; D000115:Acetylesterase / Q000737:chemistry* / Q000378:metabolism; D002236:Carbohydrate Conformation; D002384:Catalysis; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D003035:Cobalt / Q000737:chemistry*; D004789:Enzyme Activation; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D048170:Streptomyces lividans / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D006027:Glycosides; C008646:4-nitrophenyl beta-D-xyloside; D003035:Cobalt; D000115:Acetylesterase; C081851:acetylxylan esterase; C018021:cobaltous chloride