November 06, 2016
Prof. Appa Rao Podile FASc, FNASc, FNAAS,
Transglycosylation by bacterial chitinasesIndian Academy of Sciences
Annual Meeting, IISER, Bhopal
Limpens et al. Ann Rev Phytopathol. 53: 311-354, 2015
Ligand-induced composite binding groove (LysM1 and LysM3) is deeply buried in Ecp6 and displays ultrahigh
(picomolar) chitin-binding affinity, which is significantly higher than that of plant
immune receptors
(GlcNAc)8 induces dimerization of LysM1-2, and not LysM1-2 I122A
Populus trichocarpa
Rhizobium
Serratia proteamaculans
Enzyme Domainarchitecture
ChitinHydrolysis
TG Chitobiase Chitosanhydrolysis
Sp
Sp
Sp
Sp
A AChitobiaseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee A A
A A
AA
Hydrolysis/TG of Hydrolysis/TG o Sp f SpS ChiD from DP6p ChiD from DPC -P6 DP3 substrates 6-DP3 substrates DD
DP6 DP5
DP4 DP3Transglycosylation (TG) on DP6 -DP3 substrates. Monomers (DP1) were themajor end products due to chitobiase activity. DP : Degree of polymerization
Chitooligosaccharides (DP3-DP6)
TG products(DP13-DP7)
TransglycosylationN- acetyl glucosamine
Transglycosylation (TG) by Sp ChiD
Transglycosylation
Hydrolysis
Two possible mechanisms: glycoside hydrolases and transglycosidases.
Hydrolysis occurs when the glycosyl-enzyme intermediate is broken down by water.
Transglycosylation occurs when a sugar moiety acts as the glycosylacceptor.
MALDI-TOF MS analysis of products from DP3-DP6
DP3- Up to DP7
DP4- Up to DP10
DP5- Up to DP12
DP6- Up to DP13
Purushotham, P., and Podile, A.R. 2012. J. Bacteriol.
The residues targeted for mutation
Catalytic center - M226,Y228, R284, E159 andY160
Catalytic groove -F64, F125, G119, S116and W120.
Solventaccessible region-W247
A: at 30 min, B: at 360 min
Comparision of quantifiable TG products
Madhuprakash et al. J. Biol. Chem. 2012
Group Details of MutationsPosition Mutated to Out come
Group I
Catalytic center
Met -226 Ala More TG, Less hydrolysis
Tyr - 228 Ala More TG, Less hydrolysis
Glu -159 Asp Loss of both activities
Tyr - 160 Ala Only increased hydrolysis
Group II
Catalytic groove
Phe - 64 Trp More TG, Less hydrolysis
Gly - 119 Ser More TG, Less hydrolysis
Ser - 116 Gly More TG, Less hydrolysis
Phe - 125 Ala More TG, Less hydrolysis
Trp - 120 Ala No TG, More hydrolysis
Group IIISurface exposed region
Trp - 247 Ala More TG, Less hydrolysis
Entry and exit sites for SpChiD
Madhuprakash et al. 2014 BBA General Subjects
Fusion approach
CBP+ChiD
ChiD+PKDPKD+ChiD
ChiD+CBP
CBP+ChiD+PKD (CDP) PKD+ChiD+CBP (PDC)
GH18CBP21 CBP21GH18
GH18PKD GH18 PKD
CBP21 GH18 PKD PKD CBP21GH18
Madhuprakash et al. PLoS One 2015
Activity comparision of the Native and Fusion chitinase on soluble & insoluble
substrates
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Chi D Chi D+CBP21
Sp.A
ctiv
ity
-chitin-chitin
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
DA66% DA38% DA11%
Sp.A
ct.n
kat
Native chiD
Fusion chiD
Detailed structure of the novel loop Asn30-Asp42
Specific amino acid sequence that induces a novel type1-turn that changes the path of the protein chain which is
generally a -strand in the structures of other chitinases
The loop Asn30-Asp42 is internally stabilized with the helpof intra loop hydrogen bonds but, poorly connected torest of the protein.
Mutational analysis of loop Asn30-Asp42
Madhuprakash et al. Scientific Reports. 2015
HPLC-quantification profiles
Activity of SpChiD and its loop variants on chitobiose activity
No chitobiase activity for the mutants SpChiD -42 and Y28ABoth Val-35 and Thr-36 are crucial for chitobiase activity
Comparision of quantifiable TG products
DP5 increased with Y28A and deletion mutant.DP6 increased with all the loop variants and the mutant Y28A.
HPLC-quantification profiles
HPTLC and SEC analysis of products obtained from chitosan DA61%
SpSpChiDSpChiDC W114AW114A
100 mg of chitosan DA 61% substrate was used for degradation withSpChiD or W114A and purified the CHOS
Madhuprakash et al. Bioresour. Tech. 2015
FractionNumber
Type of oligosaccharides Degree of polymerizationSpChiD W114A
12 A4D7, A5D6, A6D5, A7D4 A3D8, A4D7, A5D6, A6D5 1113 A4D6, A5D5, A6D4 A3D7, A4D6, A5D5, A6D4 1014 A3D6, A4D5, A5D4, A6D3 A3D6, A4D5, A5D4 915 A3D5, A4D4, A5D3, A6D2 A3D5, A4D4, A5D3 816 A2D5, A3D4, A4D3, A5D2 A2D5, A3D4, A4D3 717 A2D4, A3D3, A4D2, A5D A2D4, A3D3, A4D2 6
MALDI-TOF-MS analysis of fractions collected through SEC
Fractions 12 to 17 generated by SpChiD and W114A, with chitosan DA61%, were analyzed through MALDI-TOF-MS
Madhuprakash et al. Bioresour. Tech. 2015
Dose-dependent elicitation of oxidative burst in rice cell suspension cultures using purified oligomeric fractions
Sp
Madhuprakash et al. Bioresour. Tech. 2015
Pe
Sm
EcPeStm
Fj
Supported by
Department of BiotechnologyMinistry of Science and TechnologyNew Delhi (TATA Innovation Fellowship)
“Nano3Bio” FP7EUROPEAN UNION
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