A plant class V chitinase from a cycad (Cycas revoluta): Biochemical characterization, cDNA isolation, and posttranslational modification

Abstract

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

The Nodulation Factor Hydrolase of Medicago truncatula: Characterization of an Enzyme Specifically Cleaving Rhizobial Nodulation Signals

“…adaptation of an enzyme to a novel microbial substrate reminiscent to receptor-ligand coevolution). In fact, class V chitinases are present in ancient land plants, including the primitive gymnosperm C. revoluta (Taira et al, 2009(Taira et al, , 2010. We suggest that MtNFH1 evolved from an ancestral defense-related chitinase and that gene duplication opened the possibility to develop a symbiotic enzyme that has the capacity to inactivate short and structurally modified NFs such as NodSm-IV(C16:2, Ac, S).…”

“…Recently, enzymatic characterization and protein crystal structure analysis were performed with the NtChiV protein expressed in Escherichia coli as well as with AtChiC, a related class V chitinase from Arabidopsis (Arabidopsis thaliana; Ohnuma et al, 2011aOhnuma et al, , 2011b. Furthermore, a class V chitinase purified from the gymnosperm Cycas revoluta exhibits transglycosylation activity at high concentrations of chitin oligosaccharides (Taira et al, 2009(Taira et al, , 2010. In addition to these enzymes, a class V chitinase-related lectin (RobpsCRA) from the legume tree Robinia pseudoacacia has been characterized.…”

Modulation of the transglycosylation activity of plant family GH18 chitinase by removing or introducing a tryptophan side chain

“…A family GH18 chitinase from the cycad, Cycas revoluta (CrChiA) is a plant chitinase to efficiently catalyze the TG reaction [10]. CrChiA hydrolyzes the substrate (GlcNAc) 4 to produce the hydrolytic product (GlcNAc) 2 as well as (GlcNAc) 3 , which was not produced directly from the initial substrate (GlcNAc) 4 , but from symmetric hydrolysis of the TG product (GlcNAc) 6 [11][12][13].…”

Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

“…Class V chitinases have a different chitin oligosaccharide degradation pattern and a big insertion compared to class III and IIIb chitinases. 9,10) The catalytic domains of plant class I, II, II-L, and IV belong to GH-19, whereas those of class III, IIIb, and V belong to GH-18. With the exception of one chitinase, plant GH-18 chitinases are only composed of one catalytic domain.…”