Progress 09/01/07 to 08/31/10
Outputs
OUTPUTS: We have disseminated the findings of this project through five publications in refereed journals, three oral presentations in American Chemical Society national meetings, and an oral presentation at a meeting in Turkey. PARTICIPANTS: Blake Mertz - formerly chemical engineering graduate student at Iowa State University and now postdoctoral research fellow in the Department of Chemistry at the University of Arizona. He is applying for many faculty jobs in chemistry and chemical engineering departments in the United States. Xun Gu - Professor of Genetics, Development, and Cell Biology at Iowa State University. Peter J. Reilly - Anson Marston Distinguished Professor in Engineering and Professor of Chemical and Biological Engineering at Iowa State University. Principal Investigator of this grant. Luis Petersen - formerly chemical engineering graduate student at Iowa State University and now researcher for OPKOS Pharmaceutical in Guadalajara, Mexico. Albert Ardevol - formerly graduate student at the University of Barcelona and now postdoctoral fellow at the Swiss Federal Institute of Technology Zurich, located in Lugano, Switzerland. Carme Rovira - researcher at the University of Barcelona. Glenn P. Johnson - formerly research technician at the USDA Southern Regional Research Center and now an information technologist at the University of Iowa. Alfred D. French - Research Chemist, USDA Southern Regional Research Center. Ian J. Barker - formerly chemical engineering undergraduate at Iowa State University. TARGET AUDIENCES: The target audience for all three results was the same: scientifically educated people in the area of utilization of agricultural products, chiefly starches and cellulosics. PROJECT MODIFICATIONS: We were originally planning to use automated docking and molecular dynamics to probe reaction mechanisms of cellulases and xylanases. However, by the time the grant funding arrived, Iowa State University's computer facilities had expanded so extensively that we were able to use quantum mechanics computations, which are much more accurate, instead. Four of the five journal publications that came from this project were done that way. We also expanded the project to work on alpha-mannosidases and cellulase-related enzymes.
Impacts
The work funded by this grant led to outcomes and impacts in three areas: 1) We showed computationally how mutations in different amino acid residues in two glycoside hydrolase families that contain cellulases and related enzymes changed the enzymes' product profiles. 2) We used quantum mechanics computation to show how different families of cellulases and amylases twisted glycosidic bonds about to be broken in different directions, depending on the family. 3) We used quantum mechanics computation again to explore the reaction mechanisms of three separate enzyme families, one of cellulases, one of alpha-mannosidases, and one of beta-xylosidases.
Publications
- Mertz, B., X. Gu, and P. J. Reilly. Analysis of Functional Divergence within Two Structurally Related Glycoside Hydrolase Families. Biopolymers, 91, 478 (2009).
- Johnson, G. P., L. Petersen, A. D. French, and P. J. Reilly. Twisting of Glycosidic Bonds by Hydrolases. Carbohydr. Res., 344, 2157 (2009).
- Petersen, L., A. Ardevol, C. Rovira, and P. J. Reilly. Molecular Mechanism of the Glycosylation Step Catalyzed by Golgi alpha-Mannosidase II. A QM/MM Metadynamics Investigation. J. Am. Chem. Soc., 132, 8291 (2010).
- Barker, I. J., L. Petersen, and P. J. Reilly. Mechanism of Xylobiose Hydrolysis by GH43 beta-Xylosidase. J. Phys. Chem. B, 114, 15389 (2010).
- Petersen, L., A. Ardevol, C. Rovira, and P. J. Reilly. Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study. J. Phys. Chem. B, 113, 7331 (2009).
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Objective 1) To computationally identify the unknown catalytic residues in the two glycoside hydrolase (GH) families with known tertiary structures but with unknown catalytic residues. We have almost solved this problem for GH family 43 (GH43) beta-xylosidases. Objective 2) To computationally determine the paths of substrate and product to and from the transition state, respectively, in one enzyme in each of the sixteen GH families with available tertiary structures. We have finished this with a GH8 endoglucanase and the work has been published by J. Phys. Chem. B. We have used quantum mechanics/molecular mechanics for this. The conformational pathway of the glycon glucosyl residue has been mapped out. We are working on a GH43 beta-xylosidase. We are finishing three other already existing projects on cellulases and xylanases with funding from this grant: 1) Measurement of functional divergence of cellulases and xylanases. This project used the program DIVERGE to determine which amino acid residues are significant to establish substrate and product specificity. An article on functional divergence in GH7 and GH16, which are very distantly related and which have the same overall tertiary structure, has been published in Biopolymers. 2) Experimental determination of the tertiary structures and kinetic properties of GH44 endoglucanases from Clostridium acetobutylicum and Ruminicoccus flavefaciens, which are very important constituents of the cellulosomes of these two organisms but which have been only very lightly studied: a) Determine the tertiary crystal structure of C. acetobutylicum endoglucanase. This work has been submitted to Applied and Environmental Microbiology. b) Determine the kinetic constants and products produced by C. acetobutylicum endoglucanse. The kinetic constants have been determined with a number of different substrates and the time courses of products made from cellotetraose, cellopentaose, and cellohexaose have been measured. Larger products predominate, suggesting that disproportionation reactions are occurring. A mathematical model to describe these reactions is being formulated. c) Determine the tertiary structures of the catalytic domain and carbohydrate binding module (CBM) of R. flavefaciens endoglucanase. Neither part of this molecule gave crystals of sufficiently high quality so that tertiary crystal structures could be determined. Determination of the CBM tertiary structure, the first for this module, by NMR is almost completely done. In addition, a complete set of binding constants for the CBM, catalytic domain, and combination of the two has been determined for a number of different substrates. 3) Twisting of glycosidic bonds of substrates by cellulases and amylases. We have produced maps of internal energies at different values of the two dihedral angles in the glycosidic bond and have placed published dihedral angles on them. In general the energies are increased by 3 to 10 kcal/mol above the energies when the glycosidic bond is in its relaxed conformation, not bound by an enzyme. The article describing this work is in press in Carbohydrate Research. PARTICIPANTS: Peter Reilly, Professor of Chemical and Biological Engineering and Anson Marston Distinguished Professor in Engineering, is the PI of this project. Blake Mertz worked on the functional divergence of GH7 and GH16, received his Ph.D. in chemical engineering in December 2008, and is now a postdoctoral fellow at the University of Arizona. Xun Gu, Professor of Genetics, Development, and Cell Biology, provided the computer program that Mertz used. Christopher Warner is working on the tertiary structures and properties of Clostridium acetobutylicum and Ruminococcus flavefaciens endoglucanases and should finish his doctoral dissertation by December 2009. Of the other coauthors of his paper, all from ISU, Julie Hoy operates the ISU X-Ray Crystallography Facility, Taran Shilling was a chemical engineering M.S. student, Michael Linnen was a chemical engineering undergraduate, Nathaniel Ginder was a biophysics graduate student, Clark Ford, Professor of Food Science and Human Nutrition, supplied the laboratory in which Warner worked, and Richard Honzatko is Professor of Biophysics. Luis Petersen conducted much of the research on the GH8 endoglucanase mechanism, with help from Albert Ardevol and Carme Rovira of the University of Barcelona in Spain. He also did much of the work on the twisting of glycosidic bonds in cellulases and amylases, along with Glenn Johnson and Alfred French of the USDA/ARS Southern Regional Research Center in New Orleans. He should finish his doctoral dissertation by December 2009. Finally, Ian Barker, a chemical engineering undergraduate, is conducting the computational study of GH43 beta-xylosidase with help from Petersen. TARGET AUDIENCES: The target audiences of this project are 1) biological and engineering researchers interested in the structures and properties of enzymes that degrade polysaccharides; 2) those who are interested in cellulose hydrolysis as part of the use of biomass and production of biofuels. PROJECT MODIFICATIONS: One of our objectives was to determine the catalytic proton donor in GH48 endoglucanases. We made no headway on this project and have abandoned it. Also, computation to determine the pathway from the substrate through the transition state to the product has proved so intensive that we will not be able to finish doing this for the majority of the cellulase and xylanase families. However, we will be able to show common patterns for both inverting and retaining GHs. On the positive side, we are using quantum mechanics/molecular mechanics metadynamics, a much more accurate computational method than the automated docking that we proposed to use, for our computation. Its use is the reason for the intensive nature of the computation.
Impacts
At this point all we can write is that the impact of the work will be shown by the many refereed articles that will come from it. In the future it is likely that some of it will lead to improvements in industrial processes.
Publications
- Mertz, B., X. Gu, and P. J. Reilly. 2009. Analysis of Functional Divergence within Two Structurally Related Glycoside Hydrolase Families. Biopolymers, 91, 478.
- Petersen, L., A. Ardevol, C. Rovira, and P. J. Reilly. 2009. Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study. J. Phys. Chem. B, 113, 7331.
- Johnson, G. P., L. Petersen, A. D. French, and P. J. Reilly. 2009. Twisting of Glycosidic Bonds by Hydrolases. Carbohydr. Res., in press.
- Warner, C. D., J. A. Hoy, T. C. Shilling, M. J. Linnen, N. D. Ginder, C. F. Ford, R. B. Honzatko, and P. J. Reilly. 2009. Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium acetobutylicum. Appl. Environ. Micobiol., submitted for publication.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Mertz, B., X. Gu, and P. J. Reilly, Analysis of Functional Divergence within Cellulase Families. 235th American Chemical Society National Meeting, New Orleans, LA. April 2008. The following talks covering material discovered in projects supported by this grant were presented: Institut fur Technische Chemie, Forchungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany, June 2008. Institut fur Technische Chemie, Technische Universitat Braunschweig, Braunschweig, Germany, June 2008. Illinois-Iowa Section, American Chemical Society, Davenport, IA, October 2008. Iowa Section, American Chemical Society, North Liberty, IA, October 2008. National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Peoria, IL, October 2008. Wichita-Duncan Section, American Chemical Society, Lawton, OK, October 2008. Department of Chemical Engineering, University of Louisiana at Lafayette, Lafayette, LA, October 2008. PARTICIPANTS: Coauthors of first paper: Blake Mertz was a doctoral student who defended his dissertation in August 2008 and will receive his Ph.D. in December 2008. He is now a postdoctoral fellow in the Department of Chemistry at the University of Arizona. Xun Gu is a Professor of Genetics, Development, and Cell Biology. Peter Reilly, the PI of this grant, is Professor of Chemical and Biological Engineering and Anson Marston Professor in Engineering at Iowa State University. Coauthors of second paper: Christopher Warner is a doctoral student who will graduate about May 2009. Taran Shilling was an M.S. student who graduated in December 2007. Michael Linnen was an undergraduate who graduated in May 2008. Clark Ford is an Associate Professor of Food Science and Human Nutrition. Peter Reilly was described above. Coauthors of third paper (not part of outputs section): Luis Petersen is a doctoral student. Albert Ardevol is a graduate student as the University of Barcelona in Spain. Carme Rovira is a researcher at the University of Barcelona. Clark Ford was described above. Wim Nerinckx is a researcher at Ghent University in Belgium. Peter Reilly was described above. Affiliations when not listed are Iowa State University. TARGET AUDIENCES: Target audiences are 1) scientists interested in structure and function of enzymes that degrade cellulose and xyans: 2) local groups of faculty, industrial personnel, and students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Three projects conducted by three doctoral students have been supported by this grant. Following are titles, authors, and abstracts of the first two journal articles to be prepared to present the material discovered in these projects: Analysis of Functional Divergence within Two Structurally Related Glycoside Hydrolase Families. Blake Mertz, Xun Gu, Peter J. Reilly. Two glycoside hydrolase (GH) families were analyzed to detect the presence of functional divergence using the program DIVERGE. These two families, GH7 and GH16, each contain members related by amino acid sequence similarity, retaining hydrolytic mechanisms, and catalytic residue identity. GH7 and GH16 comprise GH Clan B, with a shared beta-jelly roll topology and mechanism. GH7 contains fungal cellobiohydrolases and endoglucanases and is divided into five main subfamilies, four of the former and one of the latter. Cluster comparisons between three of the cellobiohydrolase subfamilies and the endoglucanase subfamily identified specific amino acid residues that play a role in the functional divergence between the two enzyme types. GH16 contains subfamilies of bacterial agarases, xyloglucosyl transferases, 1,3-beta-D-glucanases, lichenases, and other enzymes with various substrate specificities and product profiles. Four cluster comparisons between these four main subfamilies again have identified amino acid residues involved in functional divergence between the subfamilies. Kinetic properties of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum. Christopher D. Warner, Taran C. Shilling, Michael J. Linnen, Clark F. Ford, Peter J. Reilly. Clostridium acetobutylicum ATCC 824 does not grow on microcrystalline cellulose, even though it has genetic coding for a cellulosome and many cellulases. We report expression of a protein from a synthesized gene of a glycoside hydrolase family 44 endoglucanase from this strain and its subsequent purification and characterization. This previously unisolated protein is active on carboxymethylcellulose (CMC), xylan, and lichenan, slightly active on Avicel, but inactive on laminaran, mannan, and pullulan. It has kinetic efficiencies on the two xylans slightly higher than that on CMC, with an optimal activity on CMC near 55C in a short assay and an optimal pH of 5. Its energy of activation for activity is 26.9 kJ/mol and that for thermoinactivation is 230 kJ/mol. The enzyme attacks cellotetraose slowly, yielding more cellotriose than glucose and no cellobiose. At intermediate incubation times it produces mainly cellotetraose, with smaller amounts of cellobiose, cellotriose, and glucose, from cellopentaose, with cellotriose later being converted to smaller products. Cellohexaose is cleaved mainly to cellotetraose, lesser amounts of cellobiose, cellotriose, and glucose accumulating. Cellopentaose concentration peaks and then decreases. At longer incubation times only cellotriose, cellobiose, and glucose remain. These asymmetrical product distributions suggest that the enzyme catalyzes transfer reactions, and a mathematical model has been formulated to explain them.
Publications
- B. Mertz. Computational analysis of the phylogeny and thermodynamics of glycoside hydrolases. August 2008. Doctoral dissertation, Iowa State University, Ames, IA.
- The Mertz et al. manuscript above has been submitted to the journal Biopolymers. It is now under revision after receiving editor's and referees' reports. Two others are in the final stages of preparation and should be submitted by the end of January 2009.
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