Progress 10/01/01 to 09/30/05
Outputs
Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding module (Cel9A-68) followed by a fibronectin III like domain and a family 2 cellulose binding module(CBM). To elucidate its catalytic mechanism, twelve mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on carboxymethyl cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, cellotetraose, cellopentaose and 2,4-dinitrophenyl-beta-D-cellobioside. They were also tested for ligand binding, enzyme processivity, thermostability and pH optimum. The results clearly show that E424 functions as the catalytic acid, D55 and D58 act together as the catalytic base, Y206 plays an important role in binding, catalysis and processivity, while Y318 plays an
important role in binding of crystalline cellulose substrates and is required for processivity. Mutating Y429 to A reduces activity significantly but still leaves more than 20 pct of WT activity on all substrates. This is surprising since Y429 was proposed to be an essential hydrophobic platform residue. In other enzymes, mutating the hydrophobic platform residue completely inactivates the enzyme. W209 is stacked against the sugar bound in subsite -3 and it is surprising that the W209S mutant enzyme has higher than WT activity on CMC. These mutations identify two residues that are very important for Cel9A activity and processivity, H125 and W313, and one residue that is important for processivity but not for activity, F205. In addition, the results show that Y429 is probably not the hydrophobic platform for Cel9A and that W209 and 256 do not play a major role in processivity.Several amino acids located at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68 and this
enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like wild type enzyme. The FnIII like domain was deleted from Cel9A-90 reducing BMCC activity and processivity to 65 percent of the wild type. These results show that this enzyme and probably all family 9 cellulases, utalize two Asp residues for the catalytic base function, unlike all other known cellulases, which use a single residue. They also show that processivity requires tighter binding of the substrate into the active site than does the initial cleavage. Another mutation was introduced into the family 3 CBM, it changed a potential substrate binding residue from Phe to Tyr. This mutant enzyme had slightly higher activity then WT Cel9A on all tested substrates. We collaborated with a group studying a plant Cel 9 enzyme that functions in cellulose biosynthesis and showed that mutating two Trp residues in the T. fusca enzyme, which were not present in the plant enzyme, reduced its
activity to a level close to that of the plant enzyme. This work is reported in more detail in the three reported publications.
Impacts
Family 9 cellulases are the largest cellulase family and most plant and animal cellulases are in family 9. Thermobifida fusca Cel9A was the first processive endoglucanase to be identified and characterized. Similar enzymes have been found in many cellulolytic bacteria. This research provides important new information on the roles of five conserved active site residues in the mechanism of Cel9A. In addition it provided information on the factors that cause processive cleavage by this enzyme. This information will help in designing new cellulases with improved activity for the hydrolysis of the cellulose in specific biomass substrates which will improve the economics of bioethanol production.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
In this period, we have made seven new mutations in six different residues of Thermobifida fusca Cel9A and expressed all the mutant enzymes in E. coli, purified and characterized them. Their activities and extent of processivity are shown in Table I. H125 is hydrogen bonded to D58, which acts as a catalytic base along with D55. H125 clearly plays an important role in activity, as changing this residue to A gives an enzyme with less than 10 pct of WT activity on all substrates except filter paper. Mutating Y429 to A reduces activity significantly but still leaves more than 20 pct of WT activity on all substrates. This is surprising since Y429 was proposed to be an essential hydrophobic platform residue. In other enzymes, mutating the hydrophobic platform residue completely inactivates the enzyme. W209 is stacked against the sugar bound in subsite -3 and it is surprising that the W209S mutant enzyme has higher than WT activity on CMC. This may result from the smaller S
in the mutant enzyme allowing the modified substrate to fit better into the active site, but I would have thought that the loss of W209 would reduce substrate binding and thus lower activity. There is a reduction in activity on the unmodified substrates, but it is not that large. The other surprising result is that processivity is only slightly reduced by this mutation. When Y318, which hydrogen bonds to the sugar in subsite -3, was mutated, processivity was lost. We are measuring the effect of the W209 mutation on binding. We see similar results when W256, which is stacked against the sugar bound in subsite -4, is mutated to A. However, when W313, which is stacked against the sugar bound in subsite -2, is mutated to either D or G, both activity and processivity are significantly reduced. This may be due to the fact that the -2 subsite is closer to the site of cleavage than subsites -3 and -4, so that changes in this site effect the positioning of the substrate in the active site or
the mutations may cause some changes in protein structure. F205 hydrogen bonds to the sugar in subsite -2, and mutating it to A strongly reduces processivity but only slightly reduces activity. We need to determine how this mutation affects binding to see if the loss in processivity results from lowered binding. In conclusion, the properties of these new mutations identify two residues that are very important for Cel9A activity and processivity, H125 and W313, and one residue that is important for processivity but not for activity, F205. In addition, the results show that Y429 is probably not the hydrophobic platform for Cel9A and that W209 and 256 do not play a major role in processivity. In addition, mutating either of these residues to smaller AAs increases activity on CMC. Table I. Properties of T. fusca Cel9A mutant enzymes. Enzyme Activity (units/?mole) Processivity (soluble/insoluble reducing sugars) CMC S SWC BMCC Filter Paper WT 95.21 5.05 0.92 0.222 5.89 W256A 253.91 1.78
0.165 0.128 4.43 W209S 358.46 2.48 0.311 0.141 4.42 F205A 37.16 2.04 0.226 0.138 2.29 W313G 9.72 0.35 0.111 0.083 2.17 W313D 7.60 0.33 0.083 0.069 2.29 Y429A 20.87 1.19 0.295 0.131 4.05 H125A 6.48 0.23 0.062 0.049 3.00
Impacts
This research increases our understanding of the mechanism of family 9 cellulases which include most plant and animal cellulases.
Publications
- Escovar-Kousen, J.M., Wilson, D.B. and Irwin, D. 2004. Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca. Applied Biochem. & Biotech.113-116: 287-297.
- Zhou, W., Irwin, D.C., Escovar-Kousen, J. and Wilson, D.B. 2004. Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes. Biochem. 43: 9655-9663.
- Master, E.R., Rudsander, U.J., Zhou, W., Henriksson, H., Divne, C., Denman, S., Wilson, D.B. and Teeri, T.T. 2004. Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides. Biochemistry 43: 10080-10089.
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Progress 01/01/03 to 12/31/03
Outputs
Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding module (Cel9A-68) followed by a fibronectin III like domain and a family 2 cellulose binding module. To elucidate its catalytic mechanism, twelve mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned and expressed in Escherichia coli. The purified mutant enzymes were assayed for their activities on carboxymethyl cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, cellotetraose, cellopentaose and 2,4-dinitrophenyl-beta-D-cellobioside. They were also tested for ligand binding, enzyme processivity, thermostability and pH optimum. The results clearly show that E424 functions as the catalytic acid, D55 and D58 act together as the catalytic base, Y206 plays an important role in binding, catalysis and processivity, while Y318 plays an important
role in binding of crystalline cellulose substrates and is required for processivity. Several amino acids located at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68 and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like wild type enzyme. The FnIII like domain was deleted from Cel9A-90 reducing BMCC activity and processivity to 65 percent of the wild type. These results show that this enzyme and probably all family 9 cellulases utalize two Asp residues for the catalytic base function, unlike all other known cellulases which use a single residue. They also show that processivity requires tighter binding of the substrate into the active site than does the initial cleavage. Currently we are making a new set of mutants to test the role of several conserved Trp residues as well as the proposed hydrophobic platform, Tyr 429.
Impacts
This research increases our understanding of the mechanism of family 9 cellulases which include most plant and animal cellulases.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
In this period, we have constructed, expressed and characterized seven site directed mutants in four different residues of T. fusca Cel9A. Two of these, Asp55 and 58, are the residues that bind the catalytic water and mutations in them reduce Cel9A activity to about 3 percent of WT on all tested substrates, while retaining normal oligosaccharide binding. Tyr 206 was mutated to Phe and Ser, and the Phe mutation reduces activity to about 10 percent of WT with near normal binding, while the Ser mutation drasticly reduces activity and oligosaccharide binding. A Tyr 318 Ala mutation has higher activity than WT on CMC but lower activity on BMCC and it has poor oligosaccharide binding. A mutant enzyme has been constructed and expressed which lacks the fibronectin like domain which is present between the two cellulose binding domains. Cel6A activity on oligosaccharides was measured and it has very low activity on cellotetraose and high activity on cellopentose and
cellohexose. The main products of cellopentose cleavage were cellotetraose and glucose while the main products of cellohexose cleavage were cellotetraose and cellobiose. However some cellotriose was produced from all three substrates. These experiments increase our understanding of the catalytic mechanism of this interesting class of processive endocellulases. The results are very different from what we observed with the endoglucanase, T. fusca Cel 6A, where most mutations reduce activity on CMC much more then on BMCC.
Impacts
These experiments increase our understanding of this novel class of cellulase and should lead to improved enzymes for converting biomass cellulose to glucose. This would improve the economics of converting crop residues to ethanol.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The first step in this project was to model the binding of a cellulose chain into the active site and onto the surface of the attached cellulose binding domain using CHARMM. The modelling appeared to work as the chain was bound to conserved residues that had been identified as potental binding residues from the X-ray structure, Two of these, Tyr476, and Glu 424, along with two active site residues,Asp55 and Asp58 were mutated by site directed mutagenesis. The 476Phe mutant enzyme had slightly higher activity on crystalline cellulose than the wild type enzyme. We will express and characterize the other mutant enzymes, mutate other residues and start carrying out directed evolution experiments this year.
Impacts
(N/A)
Publications
- No publications reported this period
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