The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.
Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type and the other two mutants. These results suggest that, although histidines at the starch-binding site of salivary amylase are involved in starch binding and catalysis, they may not participate in Streptococcus gordonii G9B binding.
Tseng
C C
CC
Department of Oral Biology and Research Center in Oral Biology, School of Dental Medicine, State University of New York at Buffalo, 14214, USA.
Miyamoto
M
M
Ramalingam
K
K
Hemavathy
K C
KC
Levine
M J
MJ
Ramasubbu
N
N
D000409:Alanine / Q000235:genetics; D001422:Bacterial Adhesion; D001665:Binding Sites; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D005936:Glucans / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D012463:Saliva / Q000201:enzymology*; D013213:Starch / Q000378:metabolism*; D013291:Streptococcus / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000378:metabolism*
D004279:DNA, Viral; D005936:Glucans; D005960:Glucosides; D009844:Oligosaccharides; C036988:4-nitrophenylmaltoside; C031146:maltoheptaose; D006639:Histidine; D013213:Starch; D000516:alpha-Amylases; D000409:Alanine