A mutagenic study of beta-1,4-galactosyltransferases from Neisseria meningitidis.
N-terminal His-tagged recombinant beta-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant beta-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3 %). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19 %). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90 %). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27 %). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.
Park
Jae Eun
JE
Research Center for Bio-Medicinal Resources, Pai Chai University, 439-6 Doma-dong, Seo-gu, Taejon 305-600, Korea.
Do
Su-Il
SI
Lee
Ki-Sung
KS
Lee
Sang Soo
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D016296:Mutagenesis; D009237:N-Acetyllactosamine Synthase / Q000235:genetics*; D009345:Neisseria meningitidis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment
D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase; D003545:Cysteine