Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites.
Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.
Nagahashi
S
S
Department of Mycology, Nippon Roche Research Center, Kanagawa, Japan.
Sudoh
M
M
Ono
N
N
Sawada
R
R
Yamaguchi
E
E
Uchida
Y
Y
Mio
T
T
Takagi
M
M
Arisawa
M
M
Yamada-Okabe
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002687:Chitin Synthase / Q000737:chemistry*; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D002687:Chitin Synthase