Part:BBa_K3979007

Plant Chitinase Combo 1

Plant Chitinase Combo 1(PC1) is recombinant 3, Chitinase II precursor from Triticum aestivum cultivar Sumai 3, and Endochitinase 1 from Hordeum vulgare cultivar NK1558. The molecular size and weight of the protein are 1884 bp and 69.630 kDa, respectively.

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 1879
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1
  Illegal AgeI site found at 376
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1749
  

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. Purified Chitinases from Triticum aestivum cultivar Sumai 3 and Hordeum vulgare cultivar NK1558 and are well exerted broad-spectrum antifungal activity against Botrytis cinerea, Pestalotia theae, Bipolaris oryzae, Alternaria sp., Curvularia lunata, and Rhizoctonia solani. Due to the potential of broad-spectrum antifungal activity, the barley chitinase gene can be used to enhance fungal resistance in crop plants such as rice, tobacco, tea, and clover. It shows better activity in the range of body and room temperature and optimum pH around neutral value with efficient activity against a range of fungal species. Here we are utilizing chitinase parts from both these plant species to produce a combinatorial chitinase gene for more activity.

Primer Sequences Used

Fig. 1. BC1 Forward Primer

Fig. 2. BC1 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:PC1.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	-14.8	0	0
2	-14.6	20.789	30.321
3	-14.5	3.308	7.029
4	-14.3	2.898	8.011
5	-14.3	21.191	30.614
6	-14.2	3.355	7.831
7	-14	3.151	8.385
8	-14	2.015	2.781
9	-14	20.074	27.084

==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
• Singh A, Kirubakaran SI, Sakthivel N. Heterologous expression of new antifungal chitinase from wheat. Protein Expr Purif. 2007 Nov;56(1):100-9. doi: 10.1016/j.pep.2007.06.013. Epub 2007 Jul 12. PMID: 17697785.