Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

Chen, Zhiwei; Friedland, Gregory D.; Pereira, J.H.; Reveco, Sonia A.; Chan, Rossana; Park, Joshua I.; Thelen, Michael P.; Adams, Paul D.; Arkin, Adam P.; Keasling, Jay D.; Blanch, Harvey W.; Simmons, Blake A.; Sale, Kenneth L.; Chivian, Dylan; Chhabra, Swapnil R.

doi:10.1074/jbc.m112.362640

Cited by 42 publications

(41 citation statements)

References 44 publications

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“…EngD’s strong hydrolytic activity on substrates other than cellulose or beta -glucan is rare, but not unheard of with GH5 cellulases. A number of GH5 cellulases have been reported to be active on mannan, glucomannan, galactomannan, xyloglucan and xylan 44 . The ability of EngD to degrade xyloglucan, and xylan may have physiological relevance on real biomass, where cellulosomal enzymes are unable to penetrate into the cellular matrix of cellulose, hemicellulose, pectins, and other polysaccharides.…”

Section: Discussionmentioning

confidence: 99%

Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

Bianchetti

Brumm

Smith

et al. 2013

Journal of Molecular Biology

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The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising a N-terminal catalytic domain and a C-terminal carbohydrate-binding module (CBM), which is attached via a flexible linker. Here, we present the 2.1 Å resolution crystal structures of full-length EngD with and without cellotriose bound, solution small angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases the active site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products suggesting that Trp162 plays a direct role in substrate binding.

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Section: Discussionmentioning

confidence: 99%

Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

Bianchetti

Brumm

Smith

et al. 2013

Journal of Molecular Biology

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“…Nonetheless, it could be that divergence mechanisms that provide "something for nothing" may only be applicable to certain types of proteins. Enzymes in secondary metabolism (51,66,69,71), primarily in plants (22), and detoxifying enzymes, are only transiently active upon exposure to sporadic challenges and as such may remain multifunctional intermediates (73,74). Bifunctionality may also explain the facile reversal to the ancestral HSLase phenotype by merely two ancestral mutations (Table 5) (69).…”

Section: Journal Of Biological Chemistry 23923mentioning

confidence: 99%

The Evolutionary Origins of Detoxifying Enzymes

Hagit

Hugenmatter

Tawfik

2013

Journal of Biological Chemistry

115

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Background: Serum paraoxonases (PONs) comprise a family of mammalian detoxifying lactonases with different substrate specificities. Results: A newly identified bacterial PON and the reconstructed ancestor of mammalian PONs are quorum-quenching lactonases. Conclusion: PONs originated from quorum-quenching lactonases that later acquired new detoxifying functions. Significance: The evolutionary history of detoxifying enzymes may explain their ambiguous and overlapping specificity profiles.

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“…According to the evidence of GH5 plasticity that the substrate that is preferred switch one to another with only a few subtle changes . As expected for a member of GH5 subfamily 4, the activity of CelP against lichenan, barley β‐glucan, and konjac glucomannan was higher than cellobiose, methyl cellulose, and CMC, respectively.…”

Section: Discussionmentioning

confidence: 86%

Characterization of endoglucanase fromPaenibacillussp. M33, a novel isolate from a freshwater swamp forest

Kanchanadumkerng

Sakka

et al. 2016

J. Basic Microbiol.

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The newly isolated Paenibacillus sp. M33 from freshwater swamp forest soil in Thailand demonstrated its potential as a cellulose degrader. One of its endoglucanase genes from Paenibacillus sp., celP, was cloned to study the molecular characteristics of its gene product. The celP gene was recognized firstly by degenerate primer designed from Paenibacillus endoglucanase gene, and subsequently identified flanking region by inverse PCR technique. The celP gene consists of an open reading frame of 1707 bp encoding for 569 amino acids including 33-amino acids signal sequence. CelP is a member of glycoside hydrolase family 5 appended with a family 46 carbohydrate-binding module. CelP from recombinant Escherichia coli was purified by affinity chromatography. SDS-PAGE analysis of purified CelP showed a protein band at about 60 kDa. The purified enzyme gave a specific CMCase activity of 0.03 μmol min mg . It had higher activities on lichenan (0.19 μmol min mg ) and barley β-glucan (0.14 μmol min mg ). Maximum activity on lichenan was obtained at 50 °C, pH 5.0. CelP was stable over a pH range of 3.0-10.0 and retained 80% activity when incubated at 50 °C for 1 h. The properties of its CelP endoglucanase, especially substrate specificity, will make it useful in various biotechnological applications including biomass hydrolysis.

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Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

Cited by 42 publications

References 44 publications

Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

The Evolutionary Origins of Detoxifying Enzymes

Characterization of endoglucanase fromPaenibacillussp. M33, a novel isolate from a freshwater swamp forest

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