Identification and functional characterization of the NanH extracellular sialidase from Corynebacterium diphtheriae.
Corynebacterium diphtheriae, a pathogenic Gram-positive bacterium, contains sialic acids on its cell surface, but no genes related to sialic acid decoration or metabolism have been reported in C. diphtheriae. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. Interestingly, the recombinant NanH protein was secreted as a catalytically active sialidase into the periplasmic space in Escherichia coli, while the short region at its C-terminus was truncated by proteolysis. We reconstructed a truncated NanH protein (His(6)-NanH(DeltaN)) devoid of its signal sequence as a mature enzyme fused with the 6xHis tag at the N-terminal region. The purified His(6)-NanH(DeltaN) can cleave alpha-2,3- and alpha-2,6-linked sialic acid from sialic acid-containing substrates. In addition, even though the efficiency was low, the recombinant His(6)-NanH(DeltaN) was able to catalyse the transfer of sialic acid using several sialoconjugates as donor, suggesting that the reversible nature of C. diphtheriae NanH can be used for the synthesis of sialyl oligosaccharides via transglycosylation reaction.
Kim
Seonghun
S
Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon, 305-333, Korea.
Oh
Doo-Byoung
DB
Kwon
Ohsuk
O
Kang
Hyun Ah
HA
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D003353:Corynebacterium diphtheriae / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D019897:Periplasm / Q000201:enzymology*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012795:Sialoglycoproteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007527:Isoenzymes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid