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| - | == 2010 Pingry S.M.A.R.T. Team, Protein Engineering; AKR's for Biofuel Cells == | + | <StructureSection load='AdhD_with_three_cofactors_no_H.pdb' size='450' side='right' scene='2010_Pingry_SMART_Team/Adhd_final_default/2' caption=''> |
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| + | '''Return to 2010 SMART Team Page:''' [[Group:SMART:2010 Pingry SMART Team | 2010 Pingry SMART Team]] |
| - | [[Image:2010PingrySMARTTeam.jpg|frame|right|alt=alt text|(Left to Right) '''Front Row:''' Connie Wang, Edward Kong, Ed Xiao, Flo Ma, Caryn Ha, Mai-Lee Picard; '''Back Row:''' 2010 S.M.A.R.T. Team Advisor Tommie Hata, 2010 S.M.A.R.T. Team Mentor Scott Banta, Doug Ober, David Sukhin, Dylan Sun, Ricardo Vollbrechthausen, Graduate Student Elliot Campbell]]The 2010 [http://www.pingry.org Pingry School] S.M.A.R.T. Team (Students Modeling A Research Topic) is working with Dr. [http://www.cheme.columbia.edu/fac-bios/banta/faculty.html Scott Banta] and graduate student Elliot Campbell at Columbia University to learn about enzymes being engineered for use in biofuel cells. Features being engineered into these enzymes include (1) self-assembly into hydrogels, (2) alternate cofactor use, and (3) broader substrate specificity. AdhD alcohol dehydrogenase from the thermophile [http://en.wikipedia.org/wiki/Pyrococcus_furiosus ''Pyrococcus furiosus''] is one of the enzymes being engineered with these features by the Banta Lab. AdhD is a member of the aldo-keto reductase (AKR) family of oxidoreductases. Taking advantage of its innate thermostable properties, the Banta Lab is engineering AdhD for use in biofuel cells.
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| - | The logical design and engineering of AdhD is based partially on the solved structures of other enzymes belonging to the AKR family of enzymes. Structures of mutants that bind alternate cofactors and those bound to its substrate provide insight into how to engineer AdhD and other enzymes of use in a biofuel cell. The 2010 Pingry S.M.A.R.T. Team is producing physical models of various AKR's that highlight the enzymes' structural and functional characteristics that are relevant to the Banta Lab's work.
| + | '''General design guidelines''' |
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| - | {{clear}}
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| - | == What are S.M.A.R.T. Teams? ==
| + | Helix color (blue): [128,128,192] |
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| - | [[Image:CBMlogo.gif|right]][[Image:NSRR_logo.jpg|right]] "[http://cbm.msoe.edu/stupro/smart/index.html S.M.A.R.T. Teams] (Students Modeling A Research Topic) are teams of high school students and their teachers who are working with research scientists to design and construct physical models of the proteins or other molecular structures that are being investigated in their laboratories. SMART Teams use state-of-the-art molecular design software and rapid prototyping technologies to produce these unique models." -from the [http://cbm.msoe.edu/index.html MSOE Center for BioMolecular Modeling] Website.
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| - | The S.M.A.R.T. Team program was supported in part by Grant Number 1 R25 RR022749-01 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), awarded to the Center for BioMolecular Modeling.
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| | + | Sheet color (red): [255,128,128] |
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| - | == AdhD K249G/H255R double mutant and Self-assembly into hydrogels ==
| + | Three residues (ARG-R, SER-S, ASN-N; residue numbers differ by structure) that bind cofactor: display sidechains and color CPK |
| - | <applet load='AdhD_with_three_cofactors_no_H.pdb' size='400' frame='true' align='left' caption='AdhD K249G/H255R double mutant' scene='2010_Pingry_SMART_Team/Adhd_0/2'/>
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| - | <scene name='2010_Pingry_SMART_Team/Adhd_0/3'>Show NAD</scene>
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| - | <scene name='2010_Pingry_SMART_Team/Adhd_0/4'>Show NADP</scene>
| + | == AdhD K249G/H255R double mutant == |
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| - | <scene name='2010_Pingry_SMART_Team/Adhd_0/5'>Show nicotimanide</scene>
| + | '''Design description''' |
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| - | <scene name='2010_Pingry_SMART_Team/Adhd_0/2'>Remove cofactor</scene> | + | (β/α)8 secondary structure features colored blue and red. |
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| | + | Two mutated residues have backbone colored blue: K249G and H255R |
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| | + | Residues involved in cofactor binding and commonly seen in AKR's have sidechains highlighted: S251, N252, and H255R. |
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| | + | <scene name='2010_Pingry_SMART_Team/Adhd_final_default_nad/2'>Show NAD</scene> |
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| | + | <scene name='2010_Pingry_SMART_Team/Adhd_final_default_nadp/1'>Show NADP</scene> |
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| | + | <scene name='2010_Pingry_SMART_Team/Adhd_final_default_nmn/1'>Show nicotimanide</scene> |
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| | + | <scene name='2010_Pingry_SMART_Team/Adhd_final_default/2'>Return to default</scene> |
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| | + | '''Design script:''' |
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| | + | Select all |
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| | + | Backbone off |
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| | + | Cartoon off |
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| | + | Backbone 300 |
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| | + | Color white |
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| | + | Select (253-260) or (230-241) or (190-198) or (157-165) or (131-144) or (98-112) or (68-78) or (40-53) |
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| | + | Color [128,128,192] |
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| | + | Select (118-122) or (149-153) or (174-177) or (201-205) or (244-249) or (22-25) or (57-61) or (86-91) |
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| | + | Color [255,128,128] |
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| | + | Select 249 and (sidechain or alpha) |
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| | + | Wireframe 250 |
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| | + | Select 249 and sidechain |
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| | + | Color cpk |
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| | + | Select 249 and alpha |
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| | + | Color [0,0,255] |
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| | + | Select 255 and (sidechain or alpha) |
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| | + | Wireframe 250 |
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| | + | Select 255 and sidechain |
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| | + | Color cpk |
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| | + | Select 255 and alpha |
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| | + | Color [0,0,255] |
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| | + | Select (251 or 252) and (sidechain or alpha) |
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| | + | Wireframe 250 |
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| | + | Select (251 or 252) and (sidechain) |
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| | + | Color cpk |
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| | + | Select 5 |
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| | + | Color [0,0,128] |
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| | + | Select 276 |
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| | + | Color [128,0,0] |
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| | {{Clear}} | | {{Clear}} |
| - | [[2010 Pingry SMART Team Models]] | |
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| - | == Cofactor specificity == | |
| - | ==='''Modifying cofactor specificity, 2,5-diketo-d-gluconic acid reductase'''=== | |
| - | 2,5 Diketo-D-gluconic acid reductase (DKGR) is a member of the aldo keto reductase(AKR) family. 2,5-DKGR is found in corynebacterium, the genus classification of soil-dwelling bacteria, and exists in two variants: DKGR A and DKGR B. Both catalyze the reduction of 2,5 diketo-D-gluconic acid to 2Keto-L-gulonic acid, a precursor to L-absorbic acid (Vitamin C), through a series of intermediate chemical steps. Since vitamin C is an essential, main chemical manufactured worldwide, ways to increase efficiency of its production through mutations of cofactor specificity have been studied by Dr. Banta. Significantly, replacing the NADPH cofactor of 2,5-DKGR with NADH has been noted to expedite vitamin C generation because NADH is more stable, commercially less expensive, and more abundant than NADPH. Since DKGR A has a higher thermal stability at 38°C than DKGR B, mutations of this variant for increase in vitamin C efficiency have been made for adaptation to the preferable NADH cofactor. | |
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| - | <applet load='1a80' size='400' frame='true' align='left' caption='1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)' scene='2010_Pingry_SMART_Team/1a80-original/22'/>
| + | ==PDB ID: [[1m9h]], Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)== |
| - | ====PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)====
| + | <scene name='2010_Pingry_SMART_Team_Models/1m9h_final/3'>Mutant 2,5-diketo-d-gluconic acid reductase with NADH</scene> |
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| | '''Design description''' | | '''Design description''' |
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| - | 2,5-DKGR A possesses a parallel alpha-beta structural motif of the <scene name='2010_Pingry_SMART_Team/1a80-default/2'>eight alpha helices (highlighted red) and eight beta strands (highlighted blue)</scene> found in all enzymes in the aldo-keto reductase(AKR) family.
| + | (β/α)8 secondary structure features colored blue and red. |
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| - | The residue <scene name='2010_Pingry_SMART_Team/1a80-original/16'>Tyr50</scene> is found at the bottom of the active-site pocket and is conserved in all members of the AKR family. The catalytic mechanism in 2,5 DKGR A is similar to aldose reductase and other members of that super family.
| + | Four mutated residues have backbone colored orange: F22Y, K232G, R238H, A272G |
| - | The first step involves transferring a hydride ion (H-) from NADPH to the substrate leaving an oxidized cofactor.
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| - | The second step involves transferring a proton (H+) to the substrate
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| - | Tyr50 is the most likely proton donor in the catalytic mechanism, making it part of the catalytic triad.
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| - | The residues, <scene name='2010_Pingry_SMART_Team/1a80-original/17'>Ala47 and Trp77</scene>, are also found in all AKR enzymes in the active-site pocket. The active site pocket of 2,5 DKGR A is significantly smaller than the active-site pocket of human aldose reductase. The bottom of the pocket is made up of residues Phe22, Asp45, Ala47, Tyr50 (mentioned above), Lys75, Leu106, Ser139, Asn140, Trp187. The top rim of the pocket is formed by non-aromatic and apolar residues Ile49, Trp77, His108, and Trp109. The C-terminal is made up of residues Ser271 to Asp278. Ala47 and Trp77 are the only residues that are conserved in all AKR’s out of all of the active site residues. The C-terminus residues are involved in the formation of hydrogen bonds with the carbohydrate substrate as well as controlling the entry and alignment of the substrate in the active site.
| + | Other residues involved in cofactor binding: S233, V234, W187 |
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| - | Located on an extended conformation from the outer edge of the barrel is the binding site on 2,5-DKGR A for the <scene name='2010_Pingry_SMART_Team/1a80-original/12'>NADPH cofactor(shown in wireframe and colored CPK)</scene>. The NADPH cofactor is stabilized through hydrogen bonds, ionic bonds, and an aromatic pi-stacking interaction between <scene name='2010_Pingry_SMART_Team/1a80-original/21'>Trp187</scene> and the nicotinamide ring of NADPH. Although 2,5-DKGR A functions with NADPH as a cofactor, NADH is preferred for a more efficient production of vitamin C. To achieve this, mutations of the original side chains of Lys232, Phe22, Arg238, and Ala272 were conducted. Significantly, the <scene name='2010_Pingry_SMART_Team/1a80-default/1'>Lys232, Phe22, Arg238, and Ala272</scene> side chain interact with the phosphate group of NADPH. In order to accommodate for the cofactor, NADH, and the absent phosphate group, these side chains have been modified in the mutant form.
| + | Amino terminus colored dark blue: [0,0,128] |
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| | + | Carboxyl terminus colored cherry: [128,0,0] |
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| - | <scene name='2010_Pingry_SMART_Team/1a80-original/22'>Click here to revert to original display</scene>
| + | NADH shown in wireframe |
| - | {{Clear}}
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| - | <applet load='1m9h' size='400' frame='true' align='left' caption='1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH' scene='2010_Pingry_SMART_Team/1m9h_default/1'/>
| + | '''Design script''' |
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| - | ====PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)====
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| - | '''Design description'''
| + | Backbone off |
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| - | Four mutations of 2,5-Diketo-d-gluconic acid reductase have been conducted to alternate its cofcator specificity to <scene name='2010_Pingry_SMART_Team/1m9h_original/16'>NADH (shown in wireframe and colored CPK)</scene> rather than NADPH. These mutations of <scene name='2010_Pingry_SMART_Team/1m9h_original/17'>(Lys232Gly, Phe22Tyr, Arg238His, Ala272Gly).</scene>and their backbones have been highlighted orange to distinguish the change in amino acids between the 2,5-DKGR wildtype and the NADP-binding mutant.
| + | Wireframe off |
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| - | Lys 232 in the 2,5-DKGR wildtype interacts directly with the pyrophosphate group of NADPH through hydrogen bonds. However, in the 2,5-DKGR mutant,this residue has been altered into a <scene name='2010_Pingry_SMART_Team/1m9h_original/19'>Lys232Gly mutation</scene>
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| - | to adapt to the absent pyrophosphate group in NADH. Significantly, Gly lacks a side chain since no interaction is necessary due to the absent phosphate group in NADH. In addition, the mutation results in the reduction of cofactor Ka and kcat.
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| - | The <scene name='2010_Pingry_SMART_Team/1m9h_original/20'>Phe22Tyr mutation</scene> reduces the Km for both NADPH and NADH. A reduced Km creates a more efficient enzyme at a lower substrate level, therefore, improving the enzyme. The Arg238His mutation forms a pi-stacking interaction to stabilize the AKR with the cofactor. A pi-stacking interaction is extremely stable and the pi bonds are perpendicular. A common source for pi-stacking is in DNA. The <scene name='2010_Pingry_SMART_Team/1m9h_original/21'>Ala272Gly mutation</scene> mutation controls the flexibility and kinetic properties of the enzyme C-terminal tail that overlays the substrate. By increasing the speed of the C-terminal for releasing and binding, the kinetics of the substrate and cofactor's accessibility are improved significantly. The residue <scene name='2010_Pingry_SMART_Team/1m9h_original/6'>Trp187</scene> is highlighted by displaying the side chain. The pi-stacking interaction Trp187 has with the nicotinamide ring of the cofactor stabilizes the reaction.
| + | Select *a |
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| - | Not shown:
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| - | <scene name='2010_Pingry_SMART_Team/1m9h_original/23'>Ala47 and Trp77;</scene> (mentioned in P''DB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)'') are two residues that are conserved in all AKR's
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| - | The residue <scene name='2010_Pingry_SMART_Team/1m9h_original/12'>Tyr50</scene> (mentioned in ''PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)'') is a proton donor in AKR and is part of the catalytic triad conserved in all AKR's.
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| | + | Backbone 350 |
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| - | <scene name='2010_Pingry_SMART_Team/1m9h_default/1'>Click here to revert to original display</scene>
| + | Select nad |
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| - | ==='''Inherent dual cofactor use, Xylose reductase'''===
| + | Wireframe 300 |
| - | Xylose reductase is an unusual protein from the aldo-keto reductase superfamily in that the wild type is able to efficiently utilize both NADH and NADPH in its reduction of the 5 carbon sugar xylose into xylitol. Normally found in the yeast ''Candida tenuis'', it functions biologically as a homodimer unlike the majority of AKR proteins. While Dr. Banta is not actively researching this protein, Xylose Reductase's dual substrate specificity has influenced his engineering of AdhD. Because of its ability to change the conformation of two major loops, which enable different side chain conformations, Xylose Reductase can accomodate both the presence and absence of a phosphate in the cofactor.
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| - | <applet load='1K8C_chainD.pdb' size='400' frame='true' align='left' caption='1k8c, Xylose reductase with NADP+' scene='2010_Pingry_SMART_Team/1k8c_nospindefault/1'/>
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| - | ====PDB ID: 1k8c, Xylose reductase with NADP+====
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| - | Pink and blue highlight the (alpha/beta)8 barrel structure of AKR's.Cofactor (NADP+) shown in wireframe and colored CPK.
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| - | <scene name='2010_Pingry_SMART_Team/1k8c_default/12'>The residues Glu227, Lys274, Ser275, Asn276, Arg280 have sidechains shown in wireframe and colored green.</scene>
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| - | In examining this structure in relationship to 1MI3 (the same structure which uses NAD+ instead of NADP+) we can notice that many key residues interact or change their orientation to accommodate the NADP+. By examining the differences, we may be able to deduce why this efficiency is achieved and what can be altered in other Aldo-Keto Reductases to decrease cofactor specificity and increase efficiency.
| + | Color cpk |
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| - | Although it can use NADP+ more efficiently, the active site of xylose reductase has evolved to also utilize NAD+. Glu227, Asn276, and Arg280 all interact with both cofactors but in slightly different ways depending on which cofactor is present. The properties of the residues are perfect to interact with multiple key regions on the NAD+ and NADP+ molecules.
| + | Select (142-154) or (170-181) or (117-130) or (84-98) or (48-63) or (26-39) or (236-246) or (214-225) |
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| - | <scene name='2010_Pingry_SMART_Team/1k8c_default/16'>Glu227</scene> changes its interactions with the cofactor depending upon if the cofactor is NAD+ or NADP+, it has water-mediated reaction with the 3-prime alcohol group on the ribose. Similarly, <scene name='2010_Pingry_SMART_Team/1k8c_default/20'> Arg280 </scene> changes position and interacts differently with the two cofactors. <scene name='2010_Pingry_SMART_Team/1k8c_default/13'>Asn276</scene> employs hydrogen bonds with the different cofactors. The relative location on the cofacor differs in NAD+ and NADP+.
| + | Color [128,128,192] |
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| - | Lys274 and Ser275 interact only with to the NADP+ cofactor and change positions (usually turn away) when NAD+ is present.
| + | Select (18-21) or (43-45) or (71-76) or (102-107) or (133-139) or (159-163) or (183-187) or (228-231) |
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| - | <scene name='2010_Pingry_SMART_Team/1k8c_default/5'>Lys274</scene> interacts with the 2-prime alcohol group on the NADP+ ribose but turns away and does not interact when NAD+ is the cofactor. <scene name='2010_Pingry_SMART_Team/1k8c_default/22'>Ser275</scene> interacts with an oxygen on the phosphate group on the ribose of NADP+ but similarly to the Lys274, does not interact with the NAD+ cofactor.
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| - | <scene name='2010_Pingry_SMART_Team/1k8c_default/21'>Click here to revert to original display.</scene>
| + | Color [255,128,128] |
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| - | {{Clear}}
| + | Select (22 or 232 or 238 or 272) and (sidechain or alpha) |
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| - | <applet load='1MI3_chainA.pdb' size='400' frame='true' align='left' caption='1mi3, Xylose reductase with NAD+' scene='2010_Pingry_SMART_Team/1mi3_nospindefault/1'/>
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| - | ====PDB ID: 1mi3, Xylose reductase with NAD+====
| + | Wireframe 300 |
| - | As with the previous protein, pink and blue highlight the alpha and beta barrel structure common to AKR's. The cofactor NAD+ is shown in wireframe and colored CPK.
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| - | In xylose reductases' binding to NAD+, because of conformational changes on loops, <scene name='2010_Pingry_SMART_Team/1mi3_default/3'>two sidechains no longer interact with the cofactor</scene>. Lys274 and Ser275(highlighted in blue) no longer interact significantly with the NAD+. Instead, only Glu227, Asn276, and Arg280 (highlighted in green) bind to the cofactor.
| + | Select (22 or 232 or 238 or 272) and sidechain |
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| - | The <scene name='2010_Pingry_SMART_Team/1mi3_default/4'>Glu227</scene> changes so both of the oxygens on its sidechain form interactions with the 2' and 3' alcohol groups on the ribose of NAD+, while only one of the oxygens on the Glu227 interacted with the 3' alcohol group on the ribose of NADP+.
| + | Color cpk |
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| - | While xylose reductase prefers to utilize NADP+, it is able to accommodate the absence of the 2'-phosphate by adapting different conformations. <scene name='2010_Pingry_SMART_Team/1mi3_default/2'>Asn276</scene> shifts to Hydrogen bond with the hydroxy group of the NAD+ in place of the phosphate group.
| + | Select (22 or 232 or 238 or 272) and alpha |
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| - | The <scene name='2010_Pingry_SMART_Team/1mi3_default/5'>Arg280</scene> changes its configuration so an amine group on the sidechain of Arg280 can form a salt bridge with the 3' alcohol group on the NAD+, instead of forming a salt bridge with one of the oxygens on the phosphate group when the cofactor is NADP+.
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| - | The differences in the way these amino acids interact between NAD+ and NADP+ causes conformational change in the shape of Xylose reductase when used with either cofactor. <scene name='2010_Pingry_SMART_Team/1mi3_default/7'>In this image</scene>, the orange loop is Val226 through Asn229, and the yellow loop is Lys274 through Arg280. The orange loop changes shape because the different interactions between the sidechain of Glu227 (highlighted in cyan) and NAD+ as compared to NADP+, which causes the loop to bend. The yellow loop changes shape because of the changes in the way the sidechains of Asn276 and Arg280 (both highlighted in green) interact with the Nad+ as compared to NADP+. This causes the shape of the yellow loop to change, and also results in the sidechains of Lys274 and Ser275 (both highlighted in blue) to no longer interact with the cofactor NAD+, while these two do when NADP+ is the cofactor.
| + | Color [255,128,0] |
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| - | <scene name='2010_Pingry_SMART_Team/1mi3_default/1'>Click here to revert to original display.</scene>
| + | Select (233 or 234) and (sidechain or alpha) |
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| | + | Wireframe 300 |
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| | + | Select (233 or 234) and sidechain |
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| | + | Color CPK |
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| | + | Select 187 and (sidechain or alpha) |
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| | + | Wireframe 300 |
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| | + | Select 187 and sidechain |
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| | + | Color CPK |
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| | + | Select 2 |
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| | + | Color [0,0,128] |
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| | + | Select 278 |
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| | + | Color [128,0,0] |
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| | {{Clear}} | | {{Clear}} |
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| - | == Substrate specificity == | + | ==PDB ID: [[1mi3]], Xylose reductase with NAD+== |
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| + | <scene name='2010_Pingry_SMART_Team_Models/1mi3_final/7'>Xylose reductase with NAD+</scene> |
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| - | ==='''A structure of an AKR with its substrate, 3-alpha-hydroxysteroid dihydrodiol dehydrogenase'''===
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| - | A key component of Dr. Banta’s work is engineering AdhD to accept a broad range of substrates. This is a crucial component of his work, because this enzyme will be required to act upon a wide range of substrates when it is used within a practical biofuel cell. Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase demonstrates how an enzyme is specific to certain substrates and therefore help show what might be done to broaden the specificity of an enzyme. The function of this enzyme within the rat liver is to regulate / activate / deactivate steroid hormones. The enzyme does this is by reducing or oxidizing the steroid’s (testosterone) C3 ketone group. The interactions within the active site and testosterone are very specific because of the structure and positioning of the residues within the cavity. This information is important, because it will help show what might be done to AdhD to broaden its substrate specificity.
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| - | <applet load='1LWI_chainA.pdb' size='400' frame='true' align='left' caption='1lwi, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with NADP+ cofactor' scene='2010_Pingry_SMART_Team/1lwi_default/7'/> | + | |
| - | ====PDB ID: 1lwi, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with NADP+ cofactor====
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| - | Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase is abbreviated 3α-HSD.
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| - | Both NADPH (cofactor) and Testosterone (substrate, not shown) are colored CPK. NADPH can be distinguished by its orange phosphorus atoms.
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| - | <scene name='2010_Pingry_SMART_Team/1lwi_default/5'>The non-polar cavity for substrate binding is colored CPK. Leu54, Tyr55, Trp86, Phe118, Phe129, and Tyr216 are hydrophobic amino acids found in the pocket.</scene> The substrate binding pocket is non-polar because the substrate, testosterone, is a lipid and therefore hydrophobic. This is an important factor when considering how to modify substrate specificity. In Dr. Banta's fuel cell protein, the most common substrate will probably be a sugar, a hydrophilic molecule. In this case, the substrate binding pocket would be polar. | |
| | | | |
| - | The catalytic triad, which includes the most important amino acids in regards catalysis, is located at the far end of the pocket.
| + | '''Design description''' |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_cofactorbonding/1'>Orange highlights the co-factor specificity side-chains.</scene> Gln190, Asn167, Ser166 form hydrogen bonds with the nicotinamide ring in the cofactor. For more details about co-factor specificity, see the other two protein structures, which explain the subject in more depth.
| + | (β/α)8 secondary structure features colored blue and red. |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_catalytic_triad/1'>Cyan highlights the catalytic triad: Tyr55, Asp50, and Lys84.</scene> These three amino acids perform a proton relay reaction to transfer electrons between substrate and cofactor. 3α-HSD is capable of running the reaction both ways, either oxidizing or reducing the substrate and cofactor depending on the state of the testosterone. Tyr55 acts as acid, and donates a proton to the steroid-->Tyr55 forms a hydrogen bond to Lys84 for stabilization-->Lys84 forms a salt link to Asp50 for further stability.
| + | Three residues involved in NAD+ binding have backbones colored green: E227, N276, R280. |
| - | In Dr. Banta's protein, this reaction must only be run so that the sugar will be oxidized and the cofactor, reduced. The transfer of electrons from cofactor to circuit is already fairly efficient, but the key to an efficient reaction is in transferring the electron from substrate to cofactor. This is where the catalytic triad is extremely important.
| + | |
| | | | |
| - | Dark Grey highlights the beta barrel and helix structure. The barrel consists of eight parallel beta strands and eight anti-parallel alpha helices. The bottom is sealed by two antiparallel beta strands (6-10 and 13-18). This structure is common to all members of the AKR family. It provides a convenient way to keep all reactants in the same vicinity and out of the external environment. This applies to reactions in both 3α-HSD and in alcohol dehydrogenase. <scene name='2010_Pingry_SMART_Team/1lwi_betabarrel/4'>Click Here to view the Beta barrel in blue and Helices in red.</scene> The top contains two solvent exposed loops (loop A: 116-142 and loop B: 217-235)
| + | Two additional residues involved in NADP+ binding but not NAD+ binding have sidechains displayed but backbone uncolored: K274 or S275. |
| | + | |
| | + | Other residues involved in cofactor binding: S233, V234, W187, Y217 |
| | + | |
| | + | Loops that make a conformational change to bind NAD+ or NADP+ colored yellow. |
| | + | |
| | + | Amino terminus colored dark blue: [0,0,128]. |
| | + | |
| | + | Carboxyl terminus colored cherry: [128,0,0]. |
| | + | |
| | + | NAD+ shown in wireframe. |
| | + | |
| | + | |
| | + | '''Design script''' |
| | + | |
| | + | Select all |
| | + | |
| | + | Cartoon off |
| | + | |
| | + | Spacefill off |
| | + | |
| | + | Backbone 350 |
| | + | |
| | + | Color white |
| | + | |
| | + | Select nad |
| | + | |
| | + | Wireframe 250 |
| | + | |
| | + | Color cpk |
| | + | |
| | + | Select 28-41 or 55-68 or 89-103 or 147-160 or 173-182 or 201-210 or 256-266 or 278-287 |
| | + | |
| | + | Color [128,128,192] |
| | + | |
| | + | Select 19-22 or 45-49 or 77-81 or 110-112 or 165-169 or 189-192 or 213-217 or 269-271 |
| | + | |
| | + | Color [255,128,128] |
| | + | |
| | + | Select 274-280 |
| | + | |
| | + | Color [255,255,0] |
| | + | |
| | + | Select 225-229 |
| | + | |
| | + | Color [255,255,0] |
| | + | |
| | + | select (227 or 276 or 280) and (sidechain or alpha) |
| | + | |
| | + | wireframe 250 |
| | + | |
| | + | select (227 or 276 or 280) and (sidechain) |
| | + | |
| | + | color cpk |
| | + | |
| | + | select (227 or 276 or 280) and (alpha) |
| | + | |
| | + | color [255,128,0] |
| | + | |
| | + | select (274 or 275) and (sidechain or alpha) |
| | + | |
| | + | wireframe 250 |
| | + | |
| | + | select (274 or 275) and (sidechain) |
| | + | |
| | + | color cpk |
| | + | |
| | + | select (217) and (sidechain or alpha) |
| | + | |
| | + | wireframe 250 |
| | + | |
| | + | select 217 and sidechain |
| | + | |
| | + | color cpk |
| | + | |
| | + | Select 4 |
| | + | |
| | + | Color [0,0,128] |
| | + | |
| | + | Select 322 |
| | + | |
| | + | Color [128,0,0] |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_loops/1'>Purple and Blue highlight the two solvent exposed loops (Purple: Loop A, Blue: Loop B)</scene>. The loops are important for two reasons. Loop A is responsible for substrate binding. It contains many of the amino acids that create the hydrophobic substrate binding pocket. Loop B is also important to substrate binding, as it undergoes a large conformational change to accommodate the substrate. In this structure (1lwi), the substrate is absent and this loop is in its extended position. Since this loop in its extended position is in motion, an exact location can not be specified. Hence, residues Ser221 to Lys225 appear to be "missing" even though they are present in the actual protein. This opening and closing "garage door" mechanism is convenient for working through a large number of substrates, as the substrates can enter and exit easily. In the rat liver, each protein needs to convert as many steroids as possible to change the signal that is being sent out. In Dr. Banta's fuel cell, each protein would need to oxidize sugar molecules quickly to establish a current. | |
| - | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_default/7'>Revert to default scene display</scene> | |
| - | | |
| | {{Clear}} | | {{Clear}} |
| - | | |
| - | <applet load='1AFS_chainA.pdb' size='400' frame='true' align='left' caption='1afs, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone' scene='2010_Pingry_SMART_Team/1afs_default/7'/> | |
| | | | |
| - | ====PDB ID: 1afs, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone====
| + | ==PDB ID: [[1afs]], Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone== |
| - |
| + | <scene name='2010_Pingry_SMART_Team_Models/1afs_final2/1'>Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone</scene> |
| - | Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase is abbreviated as 3α-HSD. | + | '''Model description''' |
| | | | |
| - | Both NADPH (cofactor) and Testosterone (substrate) are colored CPK. NADPH can be distinguished by its orange phosphorus atoms. | |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_default/5'>The Non-polar cavity for substrate binding is colored CPK. Leu54, Tyr55, Trp86, Phe118, Phe129, and Tyr216 are hydrophobic amino acids found in the cavity.</scene> The substrate binding pocket is non-polar because the substrate, testosterone, is a lipid, and therefore non-polar. This is an important factor when considering how to modify substrate specificity. In Dr. Banta's fuel cell protein, the most common substrate will be a sugar,a hydrophilic molecule. Therefore, the substrate binding pocket must match the substrate. The catalytic triad, which includes the most important amino acids in regards to reacting with the substrate, is located at the distal, or far, end of the pocket.
| + | '''Model script''' |
| | | | |
| | + | select all |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_cofactorbonding/1'>Orange highlights the co-factor specificity side-chains.</scene> Gln190, Asn167, Ser166 form hydrogen bonds with the nicotinamide ring. For more details about co-factor specificity, see the other two protein structures.
| + | backbone off |
| | | | |
| | + | spacefill off |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1afs_safetybelt/1'>Green highlights the safety belt mechanism that is present only in 1AFS.</scene> Formed by Asp224 and Lys28, the safety belt locks the cofactor in the binding site through residues that form hydrogen bonds to the oxygens on the phosphate. This safety belt is formed when Loop B binds the substrate, and is broken upon release. As a result, this safety mechanism is present in 1AFS, but missing in 1LWI where the substrate is absent. In addition, this safety belt seems to be missing residues from Lys28 to Asp224. This is due to the fuzzy positions in the x-ray crystallography image, which leaves the exact locations of the residues unresolved.
| + | cartoon off |
| | | | |
| | + | wireframe off |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_catalytic_triad/1'>Cyan highlights the catalytic triad: Tyr55, Asp50, and Lys84.</scene> These three amino acids perform a proton relay reaction to transfer electrons between substrate and cofactor. 3α-HSD is capable of running the reaction both ways, either oxidizing or reducing the substrate and cofactor depending on the state of the testosterone. Tyr55 acts as acid, and donates a proton to the steroid. Tyr55 forms a hydrogen bond to Lys84 for stabilization. Lys84 forms a salt link to Asp50 for further stability. In Dr. Banta's protein, this reaction must only be run so that the sugar will be oxidized to reduce the cofactor. The transfer of electrons from cofactor to circuit is already fairly efficient, but the key to an efficient reaction is in transfering the electron from substrate to cofactor. This is where the catalytic triad is extremely important.
| + | backbone 350 |
| | | | |
| | + | color white |
| | | | |
| - | Dark Grey highlights the beta barrel and helix structure. The barrel consists of eight parallel beta strands and eight anti-parallel alpha helices. The bottom is sealed by two antiparallel beta strands (6-10 and 13-18). This structure is common to all members of the AKR family. It provides a convenient way to keep all reactants in the same vicinity and out of the external environment. This applies to reactions in both 3α-HSD and in alcohol dehydrogenase. <scene name='2010_Pingry_SMART_Team/1lwi_betabarrel/4'>Click Here to view the Beta barrel in blue and Helices in red.</scene> The top contains two solvent exposed loops (loop A: 116-142 and loop B: 217-235)
| + | select tes or nap |
| | | | |
| | + | wireframe 250 |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1lwi_loops/1'>Purple and Blue highlight the two solvent exposed loops (Purple: Loop A, Blue: Loop B)</scene>. The loops are important for two different reasons. Loop A is responsible for the substrate binding. It holds many of the amino acids responsible for the hydrophobic substrate binding pocket. Loop B is also important to substrate binding, as it undergoes large conformational changes to accommodate the substrate. In contrast to the previous structure, the substrate is now present (in 1afs) and this loop takes a closed position. In this closed position, residues Ser221 to Lys225 are now noticeable, since they remain fixed, as opposed to 1lwi. This opening and closing "garage door" mechanism is convenient for working through a large number of substrates, as the substrates can enter and exit easily. In the rat liver, each protein needs to convert as many steroids as possible to change the signal that is being sent out. In Dr. Banta's fuel cell, each protein would need to oxidize sugar molecules quickly to establish a current.
| + | color cpk |
| - |
| + | |
| - |
| + | |
| | | | |
| - | <scene name='2010_Pingry_SMART_Team/1afs_default/7'>Revert to default scene display</scene>
| + | select sheet |
| | | | |
| - | =='''Reference'''==
| + | select (188-191) or (162-166) or (113-115) or (81-84) or (49-52) or (19-22) or (267-270) or (212-216) |
| - |
| + | |
| - | '''Biofuel cells'''
| + | color [255,128,128] |
| - | <ref group="xtra">PMID:15669171</ref><references group="xtra"/>
| + | |
| - | <ref group="xtra">PMID:17399977</ref><references group="xtra"/>
| + | select 274-284 or 252-263 or 200-209 or 170-178 or 145-156 or 92-106 or 58-71 or 31-44 |
| - | <ref group="xtra">PMID:16781864</ref><references group="xtra"/>
| + | |
| - | '''Aldo-keto reductases'''
| + | color [128, 128, 192] |
| - | <ref group="xtra">PMID:12663943</ref><references group="xtra"/>
| + | |
| - | '''AdhD and hydrogels'''
| + | select (54 or 86 or 118 or 120 or 122 or 128 or 129 or 137 or 139 or 227) and (sidechain or alpha) |
| - | <ref group="xtra">PMID:19577577</ref><references group="xtra"/>
| + | |
| - | '''Modifying cofactor specificity, 2,5-diketo-d-gluconic acid reductase'''
| + | wireframe 250 |
| - | <ref group="xtra">PMID:9618487</ref><references group="xtra"/>
| + | |
| - | <ref group="xtra">PMID:12733986</ref><references group="xtra"/>
| + | select (54 or 86 or 118 or 120 or 122 or 128 or 129 or 137 or 139 or 227) and (sidechain) |
| - | '''Innate dual cofactor use, Xylose reductase'''
| + | |
| - | <ref group="xtra">PMID:12102621</ref><references group="xtra"/>
| + | color cpk |
| - | <ref group="xtra">PMID:12733986</ref><references group="xtra"/>
| + | |
| - | '''Substrate binding by an AKR, Rat liver 3 alpha-hydroxysteroid dihydrodiol dehydrogenase'''
| + | select (50 or 55 or 84) and (sidechain or alpha) |
| - | <ref group="xtra">PMID:8146147</ref><references group="xtra"/>
| + | |
| - | <ref group="xtra">PMID:8718859</ref><references group="xtra"/>
| + | wireframe 250 |
| - | <ref group="xtra">PMID:9261071</ref><references group="xtra"/>
| + | |
| | + | select (50 or 55 or 84) and (sidechain) |
| | + | |
| | + | color [128,0,128] |
| | + | |
| | + | select (28 or 224) and (sidechain or alpha) |
| | + | |
| | + | wireframe 250 |
| | + | |
| | + | select (28 or 224) and (sidechain) |
| | + | |
| | + | color [0,128,0] |
| | + | |
| | + | select (276 or 271 or 273 or 216) and (sidechain or alpha) |
| | + | |
| | + | wireframe 250 |
| | + | |
| | + | select (276 or 273 or 271 or 216) and (sidechain) |
| | + | |
| | + | color cpk |
| | + | |
| | + | select (24 or 166 or 167 or 190 or 216 or 219 or 221 or 270 or 271 or 272 or 276 or 273) and (alpha) |
| | + | |
| | + | color [255,128,0] |
| | + | |
| | + | select 116-142 and backbone |
| | + | |
| | + | color [0,0,255] |
| | + | |
| | + | select 217-235 and backbone |
| | + | |
| | + | color [0,0,255] |
| | + | |
| | + | Select 1 |
| | + | |
| | + | Color [0,0,128] |
| | + | |
| | + | Select 319 |
| | + | |
| | + | Color [128,0,0] |
| | + | |
| | + | |
| | + | {{Clear}} |
