Family 19 chitinase from Aeromonas sp. No.10S-24: role of chitin-binding domain in the enzymatic activity.
Family 19 chitinase from Aeromonas sp. No.10S-24 (72.6 kDa) is composed of two chitin-binding domains (ChBDs), two proline- and threonine-rich (PT-rich) linkers, and a catalytic domain. The purified enzyme was labile in a standard buffer condition and spontaneously degraded into a 46-kDa fragment upon storage at 4 degrees C. The N-terminal sequence of the 46-kDa fragment was found to correspond to the sequence of the C-terminal region of the second PT-rich linker, indicating that the 46-kDa fragment is produced by truncation of the two ChBDs and the two PT-rich linkers from the mature protein, and consists only of the catalytic domain. The hydrolytic activities toward insoluble and soluble substrates were significantly reduced by the truncation of two ChBDs. In addition, antifungal activity determined from the digestion rate of haustoria of powdery mildew was reduced by the ChBD truncation. Although the profile of the time-course of N-acetylglucosamine hexasaccharide [(GlcNAc)6] degradation catalyzed by the ChBD-truncated enzyme was similar to that of the mature enzyme protein, the specific activity of the ChBD-truncated enzyme determined from the rate of hexasaccharide degradation was lower than that of the mature enzyme. The two CBDs appear to be responsible for facilitating the hydrolytic reaction. The sugar residue affinities (binding free energy changes) at the individual subsites, (-2) (-1) (+1) (+2) (+3) (+4), were estimated by modeling the hexasaccharide hydrolysis by the mature and ChBD-truncated enzymes. The truncation of ChBDs was found to strongly affect the affinity at the (-1) site. This situation seems to result in the lower enzymatic activity of the ChBD-truncated enzyme toward the chitinous substrates.
Kojima
Miki
M
Laboratories of Enzyme System Science, Faculty of Agriculture, Kinki University, Nara 631-8505.
Yoshikawa
Tomoye
T
Ueda
Mitsuhiro
M
Nonomura
Teruo
T
Matsuda
Yoshinori
Y
Toyoda
Hideyoshi
H
Miyatake
Kazutaka
K
Arai
Motoo
M
Fukamizo
Tamo
T
D000333:Aeromonas / Q000201:enzymology*; D000595:Amino Acid Sequence; D002686:Chitin / Q000737:chemistry / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D011134:Polysaccharides; D002686:Chitin; D002688:Chitinases