Group:SMART:2010 Pingry SMART Team
2010 Pingry S.M.A.R.T. Team, Protein Engineering; AKR's for Biofuel Cells2010 Pingry S.M.A.R.T. Team, Protein Engineering; AKR's for Biofuel Cells
The 2010 Pingry School S.M.A.R.T. Team (Students Modeling A Research Topic) is working with Dr. 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 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.
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.
What are S.M.A.R.T. Teams?What are S.M.A.R.T. Teams?
"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 MSOE Center for BioMolecular Modeling Website.
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.
AdhD and Self-assembly into hydrogelsAdhD and Self-assembly into hydrogels
Cofactor specificityCofactor specificity
Modifying cofactor specificity, 2,5-diketo-d-gluconic acid reductaseModifying cofactor specificity, 2,5-diketo-d-gluconic acid reductase
2,5 Diketo-D-Gluconic acid reductase is found in corynebacterium and is part of the Aldo Keto Reductase family of enzymes. It exists in two variants: DKGR A and DKGR B; however, due to the higher thermal stability level of DKGR A, it has been chosen for mutation of cofactor specificity. 2,5 DKGR is an important enzyme in the production of vitamin C, one of the most important chemicals manufactured in the world. 2,5 DKGR does this by catalyzing the reduction of 2,5-diketo-D-gluconic acid (2,5 DKG) to 2-Keto-L-gluconic acid (2-KLG); a precursor to vitamin C. It is commercially less expensive to use NADH as a cofactor (as opposed to NADPH) and the catalyzation of 2,5 DKG into 2-KLG as well as being more abundant.
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PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)
Design description
2,5-DKGR A possesses a parallel alpha-beta structural motif of the found in all enzymes in the aldo-keto reductase(AKR) family.
The residue 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. 1. The first step involves transferring a hydride ion (H-) from NADPH to the substrate leaving an oxidized cofactor. 2. The second step involves transferring a proton (H+) to the substrate Tyr50 is the most likely proton donor in the catalytic mechanism, making it part of the catalytic triad.
The residues, , 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.
Located on an extended conformation from the outer edge of the barrel is the binding site on 2,5-DKGR A for the . The NADPH cofactor is stabilized through hydrogen bonds, ionic bonds, and an aromatic pi-stacking interaction between 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 L 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.
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PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)
Design description
Mutations of the 2,5-DKGR's conformation have been conducted for alternate cofactor specificity to rather than NADPH.
The backbone of the four residues changed between WT and NADP-binding mutant are colored orange . The is important because Gly has no side chain and there is no need for the Lys side chain to interact with a phosphate group because the phosphate group is absent in this mutation. The reduces the Km for both NADPH and NADH. A reduced Km makes 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 mutation improves the kinetic properties by making it easier for the substrate to bind with the substrate and by improving the kinetics of cofactor binding and release.
The residue is highlighted by displaying the side chain. The pi-stacking interaction Trp187 has with the nicotinamide ring of the cofactor stabilizes the reaction.
Not shown: (mentioned in PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)) are two residues that are conserved in all AKR's The residue (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
Inherent dual cofactor use, Xylose reductaseInherent dual cofactor use, Xylose reductase
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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PDB ID: 1k8c, Xylose reductase with NADP+PDB ID: 1k8c, Xylose reductase with NADP+
Pink and blue highlight the (alpha/beta)8 barrel structure of AKR's.Cofactor (NADP+) shown in wireframe and colored CPK.
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.
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.
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, changes position and interacts differently with the two cofactors. employs hydrogen bonds with the different cofactors. The relative location on the cofacor differs in NAD+ and NADP+.
Lys274 and Ser275 interact only with to the NADP+ cofactor and change positions (usually turn away) when NAD+ is present.
interacts with the 2-prime alcohol group on the NADP+ ribose but turns way and does not interact when NAD+ is the cofactor. 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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PDB ID: 1mi3, Xylose reductase with NAD+PDB ID: 1mi3, Xylose reductase with NAD+
Substrate specificitySubstrate specificity
A structure of an AKR with its substrate, 3-alpha-hydroxysteroid dihydrodiol dehydrogenaseA structure of an AKR with its substrate, 3-alpha-hydroxysteroid dihydrodiol dehydrogenase
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/ inactivate steroid hormones. The enzyme does this is by reducing 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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PDB ID: 1lwi, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with NADP+ cofactorPDB ID: 1lwi, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with NADP+ cofactor
Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase is often abbreviated to 3α-HSD.
Both NADPH (cofactor) and Testosterone (substrate) are colored CPK. NADPH can be distinguished by its by its orange phosphorus atoms.
The reason why the substrate binding pocket is non-polar is that 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 some type of sugar, which is 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 at the distal, or far, end of the pocket.
Gln190, Asn167, Ser166 form hydrogen bonds with the nicotinamide ring. For more details about co-factor specificity, see the other two protein structures.
These three amino acids perform a reaction called a proton relay 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 in which testosterone must be. Tyr55 acts as acid, donates proton to steroid-->Tyr55 forms hydrogen bond to Lys84 to stabilize-->Lys84 forms 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.
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. The top contains two solvent exposed loops (loop A: 116-142 and loop B: 217-235)
. 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 this structure (1lwi), the substrate is absent and this loop is in its extended position. 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.
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PDB ID: 1afs, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosteronePDB ID: 1afs, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone
Testosterone (substrate) and NADPH (cofactor) are colored CPK.
Leu54, Tyr55, Trp86, Phe118, Phe129, and Tyr216 are hydrophobic amino acids found in the pocket. The catalytic triad is at the distal end of the pocket.
Gln90, Asn167, Ser166 form hydrogen bonds with the nicotinamide ring.
Tyr55 acts as acid, donates proton to steroid-->Tyr55 forms hydrogen bond to Lys84 to stabilize-->Lys84 forms salt link to Asp50 for further stability
The barrel consists of eight parallel beta strands and eight antiparallel alpha helices. The bottom is sealed by two antiparallel beta strands (6-10 and 13-18).
The top contains two solvent exposed loops (loop A: 116-142 and loop B: 217-235)
ReferenceReference
Biofuel cells
- ↑ Minteer SD, Liaw BY, Cooney MJ. Enzyme-based biofuel cells. Curr Opin Biotechnol. 2007 Jun;18(3):228-34. Epub 2007 Mar 30. PMID:17399977 doi:10.1016/j.copbio.2007.03.007
- ↑ Davis F, Higson SP. Biofuel cells--recent advances and applications. Biosens Bioelectron. 2007 Feb 15;22(7):1224-35. Epub 2006 Jun 16. PMID:16781864 doi:10.1016/j.bios.2006.04.029
Aldo-keto reductases
- ↑ Sanli G, Dudley JI, Blaber M. Structural biology of the aldo-keto reductase family of enzymes: catalysis and cofactor binding. Cell Biochem Biophys. 2003;38(1):79-101. PMID:12663943 doi:10.1385/CBB:38:1:79
AdhD and hydrogels
- ↑ Wheeldon IR, Campbell E, Banta S. A chimeric fusion protein engineered with disparate functionalities-enzymatic activity and self-assembly. J Mol Biol. 2009 Sep 11;392(1):129-42. Epub 2009 Jul 3. PMID:19577577 doi:10.1016/j.jmb.2009.06.075
Modifying cofactor specificity, 2,5-diketo-d-gluconic acid reductase
- ↑ Kavanagh KL, Klimacek M, Nidetzky B, Wilson DK. Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases. Biochem J. 2003 Jul 15;373(Pt 2):319-26. PMID:12733986 doi:10.1042/BJ20030286
Innate dual cofactor use, Xylose reductase
- ↑ Kavanagh KL, Klimacek M, Nidetzky B, Wilson DK. Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases. Biochem J. 2003 Jul 15;373(Pt 2):319-26. PMID:12733986 doi:10.1042/BJ20030286
Substrate binding by an AKR, Rat liver 3 alpha-hydroxysteroid dihydrodiol dehydrogenase
- ↑ Bennett MJ, Schlegel BP, Jez JM, Penning TM, Lewis M. Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+. Biochemistry. 1996 Aug 20;35(33):10702-11. PMID:8718859 doi:10.1021/bi9604688