SASDDA6: Class I chitinase 2 from Agave tequilana (Chitinase 2, ChiAt2)

Basic information

• Entry: Database: SASBDB / ID: SASDDA6

Sample

Class I chitinase 2 from Agave tequilana

• Chitinase 2 (protein), ChiAt2, Agave tequilana

• Biological species: Agave tequilana (plant)
• Citation: Journal: FEBS J / Year: 2019; Title: A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins.; Authors: Yusvel Sierra-Gómez / Annia Rodríguez-Hernández / Patricia Cano-Sánchez / Homero Gómez-Velasco / Alejandra Hernández-Santoyo / Dritan Siliqi / Adela Rodríguez-Romero / ; Abstract: Plant chitinases are enzymes that have several functions, including providing protection against pathogens. Agave tequilana is an economically important plant that is poorly studied. Here, we identified a chitinase from short reads of the A. tequilana transcriptome (AtChi1). A second chitinase, differing by only six residues from the first, was isolated from total RNA of plants infected with Fusarium oxysporum (AtChi2). Both enzymes were overexpressed in Escherichia coli and analysis of their sequences indicated that they belong to the class I glycoside hydrolase family19, whose members exhibit two domains: a carbohydrate-binding module and a catalytic domain, connected by a flexible linker. Activity assays and thermal shift experiments demonstrated that the recombinant Agave enzymes are highly thermostable acidic endochitinases with Tm values of 75 °C and 71 °C. Both exhibit a molecular mass close to 32 kDa, as determined by MALDI-TOF, and experimental pIs of 3.7 and 3.9. Coupling small-angle x-ray scattering information with homology modeling and docking simulations allowed us to structurally characterize both chitinases, which notably show different interactions in the binding groove. Even when the six different amino acids are all exposed to solvent in the loops located near the linker and opposite to the binding site, they confer distinct kinetic parameters against colloidal chitin and similar affinity for (GlnNAc) as shown by isothermal titration calorimetry. Interestingly, binding is more enthalpy-driven for AtChi2. Whereas the physiological role of these chitinases remains unknown, we demonstrate that they exhibit important antifungal activity against chitin-rich fungi such as Aspergillus sp. DATABASE: SAXS structural data are available in the SASBDB database with accession numbers SASDDE7 and SASDDA6. ENZYMES: Chitinases (EC3.2.1.14).

Contact author

• Yusvel Sierra-Gómez (IQ, UNAM, Mexico)

Models

• Model #1962: ; Type: dummy / Radius of dummy atoms: 2.50 A / Chi-square value: 2.244 / P-value: 0.001073; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Class I chitinase 2 from Agave tequilana / Specimen concentration: 0.46-7.50
• Buffer: Name: MES 50 mM / pH: 6

Entity #1049

Name: ChiAt2 / Type: protein / Description: Chitinase 2 / Formula weight: 31.9 / Num. of mol.: 1 / Source: Agave tequilana
Sequence:

SNAQQCGSQA GGAVCPNGLC CSQFGYCGST SPYCGNGCQS QCGGGSSPTP NPPSGGGGGG GGGGSGVGSI ISSSLFDQML LHRNDAACPA NGFYTYDAFV AAANAFSGFA TTGDADTQKR EIAAFLAQTS HETTGGWPTA PDGPYSWGYC FLQEQGNPGD YCVPNDQWPC APGKKYYGRG PIQISYNYNY GPCGNAIRSD LLNNPDLVAS DPTVSFKTAL WFWMTPQSPK PSCHDVITRA WTPSAADQAA GRVPGFGVIT NIINGGVECG HGSDSRDEDR VGFYKRYCDI LGVSFGDNLD CGNQSHF

Experimental information

• Beam: Instrument name: Stanford Synchrotron Radiation Lightsource (SSRL) BL4-2; City: Menlo Park, CA / 国: USA / Type of source: X-ray synchrotron / Wavelength: 0.1127 Å / Dist. spec. to detc.: 1.8 mm
• Detector: Name: Rayonix MX225-HE

Scan

Title: Chitinase 2 from Agave tequilana / Measurement date: Apr 19, 2017 / Storage temperature: 4 °C / Cell temperature: 15 °C / Exposure time: 1 sec. / Unit: 1/A /

	Min	Max
Q	0.0067	0.3226

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 580 /

	Min	Max
Q	0.0156792	0.322595
P(R) point	1	580
R	0	97.5

Result

Type of curve: merged /

	Experimental	Porod
MW	31.9 kDa	-
Volume	-	49 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	108.4	107.28	0.13
Radius of gyration, Rg	2.51 nm	2.38 nm	0.047

	Min	Max
D	-	9.75
Guinier point	18	83