AdhD K249G/H255R double mutant and Self-assembly into hydrogels
Group:SMART:2010 Pingry SMART Team Models
(β/α)8 secondary structure features colored blue and red.
Two mutated residues have backbone colored blue: K249G and H255R
Residues involved in cofactor binding and commonly seen in AKR's have sidechains highlighted: S251, N252, and H255R.
Amino terminus colored dark blue.
Carboxyl terminus colored cherry.
Cofactor specificity
Modifying cofactor specificity, 2,5-diketo-d-gluconic acid reductase
Group:SMART:2010 Pingry SMART Team Models
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.
PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)
Design description
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.
The first step involves transferring a hydride ion (H-) from NADPH to the substrate leaving an oxidized cofactor.
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 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.
PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)
Design description
Four mutations of have been conducted to alternate its cofcator specificity to rather than NADPH. These mutations of 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.
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
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.
The 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 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 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 reductase
Group:SMART:2010 Pingry SMART Team Models
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 orientations and therefore interactions, Xylose Reductase can accomodate both the presence and absence of a phosphate in the cofactor.
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+.
Asn276 and Ser275 interact only with NADP+ cofactor. Ser275 turns away when NAD+ is present, due to a loss of phosphate interactions.
interacts with the 2-prime alcohol group on the NADP+ ribose but turns away 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.
PDB ID: 1mi3, Xylose reductase with NAD+
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.
In xylose reductases' binding to NAD+, because of conformational changes on loops, . 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.
The 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+.
While xylose reductase prefers to utilize NADP+, it is able to accommodate the absence of the 2'-phosphate by adapting different conformations. shifts to Hydrogen bond with the hydroxy group of the NAD+ in place of the phosphate group.
The 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+.
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. , 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.
Substrate specificity
A structure of an AKR with its substrate, 3-alpha-hydroxysteroid dihydrodiol dehydrogenase
Group:SMART:2010 Pingry SMART Team Models
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.
PDB ID: 1lwi, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with NADP+ cofactor
Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase is abbreviated 3α-HSD.
Both NADPH (cofactor) and Testosterone (substrate, not shown) are colored CPK. NADPH can be distinguished by its orange phosphorus atoms.
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.
Gln190, Asn167, Ser166 form hydrogen bonds with the nicotinamide ring in the cofactor. Tyr216 performs pi-stacking against the nicotinamide ring of the cofactor. For more details about co-factor specificity, see the other two protein structures, which explain the subject in more depth.
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 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. The top contains two solvent exposed loops (loop A: 116-142 and loop B: 217-235)
. 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.
PDB ID: 1afs, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone
Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase is abbreviated as 3α-HSD.
Both NADPH (cofactor) and Testosterone (substrate) are colored CPK. NADPH can be distinguished by its orange phosphorus atoms.
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.
Gln190, Asn167, Ser166 form hydrogen bonds with the nicotinamide ring in the cofactor. Tyr216 performs pi-stacking against the nicotinamide ring of the cofactor. For more details about co-factor specificity, see the other two protein structures, which explain the subject in more depth.
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.
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.
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 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.
Reference
Biofuel cells
- Barton SC, Gallaway J, Atanassov P. Enzymatic biofuel cells for implantable and microscale devices. Chem Rev. 2004 Oct;104(10):4867-86. PMID:15669171
- 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
- Khurana S, Powers DB, Anderson S, Blaber M. Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1-A resolution. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6768-73. PMID:9618487
- 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. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis. Biochemistry. 2002 Jul 16;41(28):8785-95. PMID:12102621
- 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
- Hoog SS, Pawlowski JE, Alzari PM, Penning TM, Lewis M. Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2517-21. PMID:8146147
- 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
- Bennett MJ, Albert RH, Jez JM, Ma H, Penning TM, Lewis M. Steroid recognition and regulation of hormone action: crystal structure of testosterone and NADP+ bound to 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. Structure. 1997 Jun 15;5(6):799-812. PMID:9261071
