Group:SMART:2010 Pingry SMART Team: Difference between revisions

== 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.

====PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)====

'''Design description'''

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.  

====PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)====

'''Design description'''

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>

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.  

Not shown:

<scene name='2010_Pingry_SMART_Team/1m9h_default/1'>Click here to revert to original display</scene>

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====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.

<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+.  

<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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====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.

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.

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==='''A 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 / 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====

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)

<scene name='2010_Pingry_SMART_Team/1lwi_default/7'>Revert to default scene display</scene>

 

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Both NADPH (cofactor) and Testosterone (substrate) are colored CPK. NADPH can be distinguished by its orange phosphorus atoms.

<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.

<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.

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)

<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.

   

<scene name='2010_Pingry_SMART_Team/1afs_default/7'>Revert to default scene display</scene>

=='''Reference'''==