The a-galactosidase type A gene from Aspergillus niger encodes a fully functional a-N-acetylgalactosaminidase

Natallia Kulik^a,b, Lenka Weignerová^c, Tomáš Filipi^c, Petr Pompach^d, Petr Novák^d, Hynek Mrázek^e, Karel Bezouška^d,e, Vladimír Křen^c, and Rüdiger Ettrich^a,b

^aCenter for Biotransformation and Biocatalysis, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek 136, 373 33 Nové Hrady

^bInstitute of Physical Biology, University of South Bohemia, Zámek 136, 373 33 Nové Hrady

^cCenter for Biotransformation and Biocatalysis, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4

^dInstitute of Microbiology, Laboratory of Molecular structure characterization, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4

^eDepartment of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12840 Prague 2

Keywords: a-N-Acetylgalactosaminidase / a-galactosidase type A / a-galactosidase type B / Aspergillus niger/molecular modeling/substrate binding

Two genes in the genome of from Aspergillus niger, aglA and aglB, are up to now assigned to encode for two α-galactosidases, an A and B variant. However, a careful structural analysis based on structural models of these two enzymes revealed significant differences of the active centers, which contributes to the understanding of the specificity of the hydrolyzed carbohydrates.

To confront this unexpected result with experimental data, purified protein from Aspergillus niger CCIM K2, that exhibited both α-galactosidase and α-N-acetylgalactosaminidase activity, was sequenced and could be assigned to the enzyme encoded by the gene aglA. In accordance with enzyme activity measurements, substrate docking clearly demonstrates the preference of the identified enzyme for α-D-N-acetylgalactosamine over galactose and enzyme activity measurements in the presence of thiazoline((3aR,5R,6R,7R,7aR)-(6,7-dihydroxy-5-(hydroxymethyl- 2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole) suggests for this α-N-acetylgalactosaminidase an analogous mechanism as homologous α-galactosidase.

Thus, we provide evidence that the α-galactosidase type A gene aglA from Aspergillus niger encodes in fact a fully functional α-N-acetylgalactosaminidase.

This work supported by the Academy of Sciences (AVOZ50200510, AVOZ60870520), the Ministry of Education of the Czech Republic (LC 06010, LC 7017, MSM6007665808, MSM_21620808) and by the Grant Agency of the Czech Republic (204/06/0771).