Accessory active site residues of Streptomyces sp. N174 chitosanase: variations on a common theme in the lysozyme superfamily.
The chitosanase from Streptomyces sp. N174 (CsnN174) is an inverting glycoside hydrolase belonging to family 46. Previous studies identified Asp40 as the general base residue. Mutation of Asp40 into glycine revealed an unexpectedly high residual activity. D40G mutation did not affect the stereochemical mechanism of catalysis or the mode of interaction with substrate. To explain the D40G residual activity, putative accessory catalytic residues were examined. Mutation of Glu36 was highly deleterious in a D40G background. Possibly, the D40G mutation reconfigured the catalytic center in a way that allowed Glu36 to be positioned favorably to perform catalysis. Thr45 was also found to be essential. Thr45 is thought to orientate the nucleophilic water molecule in a position to attack the glycosidic link. The finding that expression of heterologous CsnN174 in Escherichia coli protects cells against the antimicrobial effect of chitosan, allowed the selection of active chitosanase variants after saturation mutagenesis. Thr45 could be replaced only by serine, indicating the importance of the hydroxyl group. The newly identified accessory catalytic residues, Glu36 and Thr45 are located on a three-strand beta sheet highly conserved in GH19, 22, 23, 24 and 46, all members of the 'lysozyme superfamily'. Structural comparisons reveal that each family has its catalytic residues located among a small number of critical positions in this beta sheet. The position of Glu36 in CsnN174 is equivalent to general base residue in GH19 chitinases, whereas Thr45 is located similarly to the catalytic residue Asp52 of GH22 lysozyme. These examples reinforce the evolutionary link among these five GH families.
Lacombe-Harvey
Marie-Eve
ME
Département de Biologie, Centre d'Etude et de Valorisation de la Diversité Microbienne, Université de Sherbrooke, Canada.
Fukamizo
Tamo
T
Gagnon
Julie
J
Ghinet
Mariana G
MG
Dennhart
Nicole
N
Letzel
Thomas
T
Brzezinski
Ryszard
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000145:classification / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D000596:Amino Acids; D006026:Glycoside Hydrolases; C019868:chitosanase; D009113:Muramidase