Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: a study involving site-directed mutagenesis, HPLC, and real-time ESI-MS.
Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.
Taira
Toki
T
Department of Bioscience and Biotechnology, Ryukyu University, Nishihara, Okinawa, 903-0213, Japan.
Fujiwara
Maho
M
Dennhart
Nicole
N
Hayashi
Hiroko
H
Onaga
Shoko
S
Ohnuma
Takayuki
T
Letzel
Thomas
T
Sakuda
Shohei
S
Fukamizo
Tamo
T
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002688:Chitinases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D029784:Cycas / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D014312:Trisaccharides / Q000494:pharmacology
D017931:DNA Primers; D018744:DNA, Plant; D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; C054851:allosamidin; D002688:Chitinases; D000117:Acetylglucosamine