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

== AdhD K249G/H255R double mutant and Self-assembly into hydrogels ==

(β/α)8 secondary structure features colored blue and red.  

Two mutated residues have backbone colored blue: K249G and H255R  

Amino terminus colored dark blue.

Carboxyl terminus colored cherry.

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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.

====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>

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

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==='''Inherent dual cofactor use, Xylose reductase'''===

 

====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.

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== Substrate specificity ==

==='''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====

Both NADPH (cofactor) and Testosterone (substrate, not shown) are colored CPK. NADPH can be distinguished by its orange phosphorus atoms.

====PDB ID: 1afs, Rat liver 3-alpha-hydroxysteroid dihydrodiol dehydrogenase with cofactor and testosterone====  

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_default/7'>Revert to default scene display</scene>

=='''Reference'''==