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