Part:BBa_K3979006

H. vulgare Wild-Type Chitinase

Endochitinase 1 derived from Hordeum vulgare cultivar NK1558. The molecular size and weight of the protein are 33.40 kDa and 981 base pairs.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1
Illegal BamHI site found at 7
Illegal XhoI site found at 976
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1
Illegal NgoMIV site found at 88
1000
COMPATIBLE WITH RFC[1000]

Overview

Chitinases are glycosyl hydrolases (GH) whose catalytic mechanism involves the hydrolysis of the β-1-4-linkage in the N-acetyl-D-glucosamine polymer of chitin, which is a major structural component of fungi. Chitinases are classified into two types based on their cleavage and hydrolysis mechanisms: endochitinase and exo-chitinase. Plants lack chitin, and it has been proposed that plant chitinases play a role in defense response by degrading chitin in invading pathogens’ cell walls. Chitinases isolated from plants have been shown to inhibit the growth of chitin-containing fungi in-vitro and in-vivo, and over-expression of chitinases in plants confers resistance against a variety of fungal pathogens. Chitinases are classified into seven classes (Class I–VII) and contain catalytic domains that define the two major GH families (GH18 and GH19). GH18 chitinases (class III and V) are found in a wide range of organisms, while GH19 chitinases (class I, II, IV, VI, VII) are found mainly in higher plants and are responsible for the majority of chitinolytic activity^[1].Chitinase genes code for enzymes that degrade chitin polysaccharides from their reducing end. Plants are significant sources of chitinase proteins where they use these enzymes to degrade chitin for nutrition. By screening a barley genomic library, a class I chitinase clone 6N was isolated. In the present investigation, the barley chitinase gene was subcloned, overexpressed in Escherichia coli BL-21 (DE3) and purified chitinase was evaluated for in vitro antifungal activity against the major phytopathogenic fungi^[2].

Primer Sequences Used

Fig.1. Barley Forward Primer

Fig. 2. Barley Reverse Primer

Protein Structure from RaptorX

The annotated sequence was input into the RaptorX server to give us the predicted 3D structure in the form of a PDB file.
File:Barley.mp4

Autodock Results

The receptor, here, is our engineered chimeric chitinase and the ligand is the Chitin octamer(CID 24978517). The threshold binding energy is -6kcal/mol which is generally accepted as the cut-off in ligand-binding /docking studies, any value more negative than this is considered significant. So, this protein will show binding with the chitin polymer. The protein structures were prepared before docking by removing water molecules, adding polar hydrogen atoms, and adding Kollman charges. A grid box was created so as to eliminate any surface binding and provide us with better and more reliable results. These modifications are necessary for the efficient binding of the ligand to the protein through non-covalent interactions.

Mode	Affinity (kcal/mol)	Dist from RMSD L.B.	Best mode RMSD U.B.
1	-11.0	0	0
2	-10.7	6.118	10.501
3	-10.7	6.248	11.334
4	-10.5	5.583	10.811
5	-10.4	19.129	24.774
6	-10.4	5.812	10.986
7	-10.4	4.083	13.453
8	-10.3	7.474	13.770
9	-10.2	3.775	11.389

==References==

Bartholomew ES, Black K, Feng Z, et al. Comprehensive Analysis of the Chitinase Gene Family in Cucumber (Cucumis sativus L.): From Gene Identification and Evolution to Expression in Response to Fusarium oxysporum. Int J Mol Sci. 2019;20(21):5309. Published 2019 Oct 25. doi:10.3390/ijms20215309
Kirubakaran SI, Sakthivel N. Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli. Protein Expr Purif. 2007 Mar;52(1):159-66. doi: 10.1016/j.pep.2006.08.012. Epub 2006 Sep 6. PMID: 17029984.