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

==='''Inherent 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.

 

<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.

<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.

<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.

   

=='''Reference'''==