Center for Biocatalysis and Biotransformation, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek 136, CZ37333 Nové Hrady, Czech Republic.
Glycobiology. 2010 Nov;20(11):1410-9. doi: 10.1093/glycob/cwq105. Epub 2010 Jul 4.
Two genes in the genome of Aspergillus niger, aglA and aglB, have been assigned to encode for α-d-galactosidases variant A and B. However, analyses of primary and 3D structures based on structural models of these two enzymes revealed significant differences in their active centers suggesting important differences in their specificity for the hydrolyzed carbohydrates. To test this unexpected finding, a large screening of libraries from 42 strains of filamentous fungi succeeded in identifying an enzyme from A. niger CCIM K2 that exhibited both α-galactosidase and α-N-acetylgalactosaminidase activities, with the latter activity predominating. The enzyme protein was sequenced, and its amino acid sequence could be unequivocally assigned to the enzyme encoded the aglA gene. Enzyme activity measurements and substrate docking clearly demonstrated the preference of the identified enzyme for α-N-acetyl-d-galactosaminide over α-d-galactoside. Thus, we provide evidence that the α-galactosidase type A gene aglA from A. niger in fact encodes a fully functional α-N-acetylgalactosaminidase using a retaining mechanism.
黑曲霉基因组中的两个基因 aglA 和 aglB，被分配到编码α-d-半乳糖苷酶变体 A 和 B。然而，基于这两种酶的结构模型对其一级结构和 3D 结构的分析表明，它们的活性中心存在显著差异，这表明它们对水解碳水化合物的特异性存在重要差异。为了验证这一意外的发现，我们对 42 株丝状真菌的文库进行了大规模筛选，成功地从黑曲霉 CCIM K2 中鉴定出一种同时具有α-半乳糖苷酶和α-N-乙酰半乳糖胺酶活性的酶，后者活性占主导地位。对酶蛋白进行测序，并对其氨基酸序列进行了明确的归属，该酶被编码为 aglA 基因。酶活性测定和底物对接清楚地表明，鉴定出的酶对α-N-乙酰-d-半乳糖胺苷的偏好超过了α-d-半乳糖苷。因此，我们提供了证据表明，黑曲霉中的α-半乳糖苷酶 A 型基因 aglA 实际上通过保留机制编码了一种具有完全功能的α-N-乙酰半乳糖胺酶。
