5oa5

CELLOBIOHYDROLASE I (CEL7A) FROM HYPOCREA JECORINA WITH IMPROVED THERMAL STABILITY

spinqualitylabelsall models 5oa5, resolution 2.10Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5oa5 is a 2 chain structure. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: GOL, NAG NonStd Res: PCA Activity: Cellulose 1,4-beta-cellobiosidase (non-reducing end), with EC number 3.2.1.91 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function [GUX1_HYPJE] The biological conversion of cellulose to glucose generally requires three types of hydrolytic enzymes: (1) Endoglucanases which cut internal beta-1,4-glucosidic bonds; (2) Exocellobiohydrolases that cut the dissaccharide cellobiose from the non-reducing end of the cellulose polymer chain; (3) Beta-1,4-glucosidases which hydrolyze the cellobiose and other short cello-oligosaccharides to glucose. Publication Abstract from PubMed Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 degrees C. Enhanced thermal stability is desirable to enable use of higher process temperatures and improve the economic feasibility of industrial biomass conversion. Here, we have enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which has a 10.4 degrees C increase in Tm and a 44-fold greater half-life compared to the wild type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 degrees C. The X-ray crystal structure of the catalytic domain was determined at 2.1 A resolution and shows that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations reveal the catalytic domain of wild type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, while at elevated temperature (475 K and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites. Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.,Goedegebuur F, Dankmeyer L, Gualfetti P, Karkehabadi S, Hansson H, Jana S, Huynh V, Kelemen BR, Kruithof P, Larenas EA, Teunissen PJM, Stahlberg J, Payne CM, Mitchinson C, Sandgren M J Biol Chem. 2017 Aug 31. pii: jbc.M117.803270. doi: 10.1074/jbc.M117.803270. PMID:28860192 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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