User:Sharwat Jahan/Sandbox1 Desaturase

From Proteopedia

Abstract

By facilitating the introduction of double bonds by the removal of two hydrogen atoms, desaturase enzymes have proven to be significant in introducing fatty acid functionality and diversification. Manipulating desaturases by filling in the active site would allow the position at which double bonds are formed on saturated fatty acids to be changed. As a result, the functionality of the unsaturated fatty acid product can be improved. Modifying the desaturase can also change the enzyme’s specificity to its substrate allowing it to bind a wider variety of fatty acid chain lengths. A desaturase that can bind to several fatty acids with different chain lengths would be more useful commercially. Through the use of Rasmol, a 3-D molecular visualization software, 18:0 (18 carbon long) fatty acid substrate of the castor desaturase is modeled with its di-iron active site with its six ligands and an 18:0 fatty acid substrate. The di-iron active site is represented as orange in color embedded within a core helical bundle labeled light blue while the non-helical shown in white. Positioned adjacent to this di-iron active site, the 18:0 fatty acid substrate is designated in gray. There are six ligands in the active site surrounding the substrate and di-iron center (two histidines- and , and four glutamic acids- , , , and ). These amino acid residues help coordinate the active site and the correct positioning of the ligands are important for the reactivity of the di-iron center. His146, His232, Glu105, Glu143, Glu196, and Glu229 are all in the CPK color scheme. If manipulated successfully, this enzyme may be able to bind a 16:0 or a 14:0 fatty acid substrate rather than the just the normal 18:0, as well as placing double bond between two carbons other than the 9 and 10 carbons. This research on castor desaturase can use genetically altered plants with modified desaturase enzymes to serve as “green-factories” which can produce specific renewable resources as well as bioenergy sources. Consequently, this may help to meet the world’s rising demands on natural resources. A 3-D Model of this desaturase would be built by the Rapid Prototype Facility at Milwaukee School of Engineering (MSOE). This 18:0 fatty acid and the diiron active site model would present a physical representation to better understand the structure and orientation of the key elements within this desaturase model.

Introduction

Desaturase enzymes facilitate the addition of double bonds at the expense of two hydrogen atoms (a dehydrogenation reaction). Δ9 stearoyl-acyl carrier protein (ACP) desaturase is a μ(single)-oxo-bridged di-iron enzyme that specifically places a cis-double bond at the C-9 and C-10 position of stearoyl-ACP. This dehydrogenation requires molecular oxygen that is activated by the enzyme’s diiron active site which is coordinated by six ligands: , , , , , and . Derived from the castor oil plant Ricinus communis, Δ9 desaturase is formed by the connection of stearic acid to ACP via a thioester linked to a pantetheine group. ACPs serve as essential cofactors in fatty acid synthesis especially in plants and bacteria.

Methods

Rasmol is a 3-D molecular visualization software that allows the modeling of the desaturase and placing of emphasis on specific aspects of the molecule such as the secondary structure, amino acids, and hetero-groups. Rasmol reads a “.pdb” file of the molecule and saves the modifications as a “.spt” file. For the desaturase of interest, focus is placed on the 18:0 fatty acid substrate, the di-iron active site with its six ligands and the 4 helical bundle. The format used to model the desaturase is in standard Rasmol dimensions. These include backbone 300” with “wireframe off,” the di-iron active site along withits six ligands are modeled in “spacefill 275” and monitor lines in “spacefill 200”. Furthermore, the backbone is color-coded gray with the helices emphasized in blue. The fatty acid substrate, the di-iron site and the six ligands are color-coded in standard CPK colors: oxygen –red, nitrogen - blue, carbon - gray, and iron - orange .Monitor lines, not part of the actual molecule, are dispersed throughout the backbone, shown in white, with the purpose of supporting fragile regions of the structure when it is printed into a3-D physical model. To print it into a physical model, the file initially in “.spt” format is converted to “.stl” format and sent to the Milwaukee School of Engineering (MSOE), where it is printed using the ZCorp 510, a rapid prototyping machine.

• Note: For purposes of clarity, the scene of the molecule show the 18:0 fatty acid substrate in purple instead ofstandard CPK colors.

Model of Structure

The diiron active site (orange) is embedded within a four-helical bundle (light blue) while non-helical portions are indicated in white. A deep, narrow hydrophobic cavity along with the 18:0 fatty acid substrate (gray) are positioned adjacent to this active-site diiron cluster. Coordinating the diiron active site and facilitating its reactivity, ligands , , , , , and are shown in CPK color scheme. At the resting state of the enzyme, the the two irons of the active site are coordinated by 5 ligands each. However in the presence of the substrate stearoyl-ACP, there is a carboxylate shift of Glu-196 away from Fe-2. This results in Fe-2 to be coordinated by four ligands instead of five. This carboxylate shift increases the enzyme’s reactivity against molecular oxygen. This reduced ferrous center with molecular oxygen leads to a peroxo intermediate. This is followed by a highly reactive intermediate that is able to remove hydrogen atoms from the saturated fatty acid bound in the active site.

Proposal

Desaturases like the stearoyl-ACP desaturase are highly specific to the position at which they form the double bond on their substrate. If the desaturase could be modified to form a double bond at different locations, then it may create a product that is functionally improved and hence much more commercially valuable. To modify desaturase, you can “fill up” the active site by substituting smaller amino acids with larger amino acids of similar properties. By using amino acids with similar properties, the function of the enzyme is maintained. For instance, if or , both small, nonpolar amino acids, are substituted with Phe and Trp, which are large non polar amino acids, the active site would be partially filled. This would consequently prevent the substrate from fully entering the active site. Furthermore, this would cause a change in the position of where the double bond is formed on the fatty acid. Similarly, if , a relatively small polar amino acid, is substituted with Gln, a larger polar amino acid with similar pKa, it would also help block the substrate from completely entering the active site. These types of substitutions can be used to modify the desaturase and form double bonds on different positions of the substrate. To obtain these modified desaturases, plants can be genetically altered to then produce them for the commercial needs of a growing population in need of renewable resources.

Conclusion

Desaturase enzymes have proven to be significant in fatty acid functionality and diversification. By modifying these castor desaturases, improvement in fatty acid product and enzymatic binding to a wider variety of fatty acid chain lengths may come about if manipulated successfully. Specifically, placement of the double bond between two carbons other than the 9 and 10 carbons, and the enzymatic binding of a 16:0 or 14:0 fatty acid substrate rather than the normal18:0. Research on castor oil desaturase can be used to genetically alter plants to serve as “green-factories” which can produce specific oils. This provides us with a way to produce renewable resources as well as sources of bioenergy. If successful, these “green-factories” can replace the enormously costly and environmentally harmful chemical factories with fields of plants. These fields would be much more cost-efficient and eco-friendly, thus creating a bridge between our economy and our earth.