Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4.
Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.
Puchart
Vladimír
V
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, 531 boulevard des Prairies, Laval, Québec, Canada H7V 1B7. chempuk@savba.sk
Gariépy
Marie-Claude
MC
Shareck
François
F
Dupont
Claude
C
D000115:Acetylesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D017124:Conserved Sequence; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D048170:Streptomyces lividans / Q000201:enzymology*
D001426:Bacterial Proteins; D006639:Histidine; D001216:Asparagine; D000115:Acetylesterase; C081851:acetylxylan esterase