| Hydrolases, especially glycoside hydrolases and ester hydrolases, have beenwidely used in many industrial processes, including food, feed, detergen, biofuels,pharmaceutical and environmental industries, accounting for more than80%of allindustrial enzyme sales. After decades of research, much information about theircatalytic mechanism has been accumulated. However, a comprehensive understandingof their substrate specificity and thermal stability is still lack, which makes it difficultto design and engineer these enzymes. Resolving their three-dimensional structurescould provide more accurate information regarding the structure-function relationshipand facilitate the design of an ideal enzyme. Based on X-ray crystallographic analysis,herein we performed the following research on several representative glycosidehydrolases and ester hydrolases.(1) Structural and biochemical analysis of a thermostable xylanase CbXyn10BXylanases efficiently cleave the β-1,4-backbone of xylan and play an importantrole in the degradation and utilization of this important bioresource. In this section, wecloned, overexpressed and purified a GH10xylanase, CbXyn10B, fromCaldicellulosiruptor bescii. The recombinant enzyme is highly active towards xylansand highly thermostable, with specific activity of about450U mg-1and a half-life ofabout7.7h at60°C. To investigate the structural basis for its remarkable properties, wedetermined the crystal structures of CbXyn10B in its native form and in complex withxylobiose and xylotriose at2.05,1.7and1.9resolutions, respectively.CbXyn10B displays a typical (β/α)8-barrel fold. The sidechains of Lys306and Arg314on surface loop8form several hydrogen bonds with carbonyl groups on the mainchain of loop8(Phe309and Pro310) and loop7(Phe255, Asp257and Arg259)through three conserved waters. This extensive hydrogen bond network betweensurface loops might play an important role in maitaining CbXyn10B stability. Besides, the N-and C-terminal of the enzyme tightly contact with each other through anaromatic cluster consisting of Tyr17, Phe20, Phe21and Phe337, and cation-πinteractions of Phe20-Arg288and Lys16-Phe337-Arg285, which might also contributeto CbXyn10B thermostability. Alanine scanning of the four key residues resulted insignificantly decreased thermostability of the mutants (K306A, R314A, F20A, F337Aand F20A/337A), indicating that the interactions mentioned above are critical forCbXyn10B thermostability.(2) Biochemical characterization of a thermostable xylanase CbX and functionalanalysis of its structural modulesGH11xylanases are a family of well characterized glycoside hydrolases. Manymembers of this family consist of a catalytic domain (CD) as well as at least onecarbohydrate binding module (CBM). To further investigate the effect of non-catalyticdomains on GH11xylanases, we cloned a GH11xylanase, CbXyn11A, from C. bescii(CbX for short) and its truncated mutants. All CbX and its derivatives weresystematically characterized. CbX is highly active and highly thermostable, withspecific activities of about2500U mg-1and a half-life of about9.5h at70°C.Compared to CbX, mutants CbX-CDL and CbX-CD with the deletion of CBM, show33-and25-fold increase in thermostability, respectively, suggesting that CBM plays animportant role in regulation of enzyme thermostability. Besides, sequence ananlysisshowed that the C-terminus of CbX-CD is about5amino acids longer than manyhomologues. The5amino acids deletion mutant (CbX-CDΔC5) displayed significantly(60-fold) decrease in thermostability compared to CbX-CD. Analysis of the structuremodel of CbX-CD showed the deletion disrupted the abundant hydrogen bonds withresidues around. These results provide important information for understanding of thethermostability mechanism of GH11xylanases and contribute to engineer theseenzymes.(3) Crystal structure of a thermostable cellobiohydrolase CbCBH48AGH48cellobiohydrolases can work synergistically with many cellulases for thedegradation of lignocellulose. Therefore, they are one of critical components ofnumerous natural lignocellulose-degrading systems. At present, only five structures ofGH48cellobiohydrolases have been reported and very limited knowledge for theircatalytic mechanism and structure-function relationship has been known. Our previousresearch showed a GH48cellobiohydrolase from C. bescii, CbCBH48A, was highlythermostable and can synergistically increase the activity of endo-beta-1,4-glucanases from C. bescii (CbCel9A) and F. nodosum Rt17-B1(FnCel5A). Here, we determinedthe structures of CbCBH48A and the enzyme-substrate complexes by X-ray (1.70-2.00resolutions). Its structure consists of an (α/α)6-helix barrel with long loops on theN-terminal side of the inner helices, which form a tunnel, and an open cleft regioncovering one side of the barrel. The active center is located in a depression at thejunction of the cleft and tunnel regions. Glu60is the proposed proton donor in thecleavage reaction, while the corresponding base is proposed to be Asp231. Bothsubstrates cellobiose and cellotetraose were enclosed in the cleft, with thenon-reducing end towards the active center, indicating that CbCBH48A cleavesprocessively cellobiose from the reducing to the non-reducing end of the cellulosechain. The structures we obtained enriched the structural information for exploring thestructure-function relationship of GH48cellobiohydrolase.(4) Structural basis for the regulation of substrate specificity of a thermstablelactonase GkaPPhosphotriesterase (PTE) can efficiently hydrolyze organophosphorus compounds(OPs) and has important potential applications in the biodegradation of OPs, theprevention and treatment of animal organophosphorus poisoning and the detection ofOPs in biosensor. A posphotriesterase-like lactonase, GkaP, from G. kaustophilusHTA426was highly thermostable, with a half-life of about8h at80°C. And it canpromiscuously hydrolyze several OPs. To further probe the structural basis for itsspecificity and improve its activity towards OPs, we analyzed the squences andstructures of GkaP and its homologues in both PLL and PTE families. We foundresidue at99position in GkaP active center may contribute to the enzyme specificity.For mutant Y99L, the catalytic efficiency towards paraoxon increased by11-fold; onthe contrary, its catalytic efficiency towards decalatone decreased by15-fold, leadingto a165-fold switching in specificity. To illustrate the structure-function relationship,we determined the structures of GkaP and mutant Y99L at1.60and1.75resolution, respectively. Structure analysis showed that residues at99position regulatethe conformation of loop7, which is near the active site. In the wide-type GkaP, theloop7displays a closed conformation; but this loop shifts6.6-8.6away from activecenter in Y99L and displays an open conformation. The size of the binding pocket inY99L greatly increases, thereby facilitates the substrate binding and product release,leading to increased activity toward ethyl-paraoxon.(5) Structural basis for the regulation of stability of C. antarctica lipase B Candida antarctica lipase B displays strict enantioselectivity and is one of themost widely used lipases in fine chemical industry. However, the lack ofthermostability hampers its wide application. In our previous study, we demonstratedthat kinetic stability of CalB can be enhanced by mutating the structurally flexibleresidues within the active site. The mutants D223G, L278M, and D223G/L278Mexhibited3-,6-and13-fold increase in thermostability, respectiviely. To explore thestructural basis for the enhanced thermostability, we determined the structures of CalBand its mutants D223G, L278M and D223G/L278M (1.50-1.60resolutions).Structure analysis showed that the mutations in D223G/L278M led to the formation ofa new hydrogen bond network with the268-281segment (containing the α10helix),which increased the rigidity of this segment and enhanced the enzyme kinetic stability.The result may provide an new method for improving enzyme stability. |
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