Karen Bulaklak/Sandbox1 Glucosamine 6 Phosphate Synthase

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== Abstract ==
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== Discussion ==
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C. albicans is a fungus normally present on the skin and within the mucous membrane. Despite its common presence, any stimuli for overgrowth can induce the invasion of C. albicans into the throat, intestines, and heart valves as it travels down the bloodstream. The fungus can be present in various morphologies such as rounded buds (yeast), pseudohyphae, and hyphae (mycelia). However, it is the hyphal form that induces the invasion into tissue. Glucosamine 6 phosphate (GlcNAc) synthase is an enzyme that catalyzes the first step in the pathway that ultimately results in hyphal formation. As a result, UDP-GlcNAc, a precursor of chitin, is generated. However, the specific mechanisms for the production of UDP-GlcNAc by this enzyme are not clearly understood.
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A 3-dimensional model of Glucosamine-6-phosphate synthase was created highlighting vital features of the protein’s isomerase domain:
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Eukaryotic GlcNAc synthase is a dimer of two dimers and displays a tetrameric structure. Each subunit is composed of two domains, GAH and ISOM. GAH is involved in glutamine hydrolysis while the ISOM domain is directly involved with the isomerization of fructose 6 phosphate to glucose 6 phosphate. We have analyzed and constructed a 3D physical model of GlcNAc synthase that focuses on the ISOM domain (346-712 aa) in complex with UDP-GlcNAc and fructose 6 phosphate using the computer software RasMol. The secondary structure of each subunit involves beta sheets (light pink) with hydrogen bonds (white) that provide stabilization to the model. In addition, we have selected residues involved in the tetramerization of this enzyme, an intermolecular interaction that is not observed in prokaryotes. Our physical model depicts residues involved in tetramerization (524-527). Other contacts related to tetramerization between the subunits are present in residues 391-445 (yellow). We have also selected fructose 6 phosphate (light green) in complex with the enzyme. Residues of the enzyme that are in close proximity to this molecule; Glu591, Lys588, His607 (light blue) reveal a possible binding region. Since the experimental procedures involved the replacement of GlcN-6P with Fructose-6P, and the combination of the obtained crystal structures with and without Fructose-6P using computer software, we only see the presence of one Fructose-6P ligand binding despite four possible binding sites. Finally, UDP-GlcNAc (magenta) molecule and surrounding amino acids 474-492 (royal blue) are represented to highlight the binding pocket. Although UDP-GlcNAc is present in complex only on chains A and B, the binding pocket is evident on all four chains. This is due to the fact that there were two crystal structures obtained for the synthase. Crystals were grown both in the absence of UDP-GlcNAc and in the presence of UDP-GlcNAc (Konariev et all 2007). Both crystal structures were used to manually form the final model within our referred PDB structure (2PUT) using computer software, and thus we see the UDP-GlcNAc binding in one chain, while the other dimer chain of each pair is free of the ligand.
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*The UDP-GlcNAc binding pocket, colored in dark blue, includes the sidechains Gly474, Val476, Ser484, Thr487, His492 and residues 489-491.
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**Coordinates with metal cation (in red) when substrate (in magenta) is bound, possibly indicating that a positive charge is needed to increase the stability of the enzyme-substrate complex.
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*Fructose-6-phosphate, colored in green, interacts with sidechains of Glu591, Lys588, His607 (shown in light blue). These residues form a small binding domain for F6P, which has yet to be completely characterized.
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*These two important binding sites appear on opposite sides of each chain, perhaps indicating that the substrates do not greatly effect each others’ interaction with the protein.
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*Tetramerization sites create a horizontal line of symmetry across the protein, separating identical subunits (A and D, B and C), a so-called “dimer of dimers.”
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*Beta sheets are also shown in light pink, which could provide internal support to the protein.
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Although the distinct tetramerization, fructose interaction sites, and UDP-GlcNAc binding pocket selected in the physical model represent important areas in the development of the invasive mycelia form of C. albicans, there are numerous other factors such as phosphorylation sites present on the GAH domain, amino acids involved in amido transfer, and other regions which are also highly significant. Further investigation could reveal possible competitive inhibition for the binding region of fructose and tetramerization sites which can the synthase and thus prevent the formation of UDP-GlcNAc and ultimately inhibiting mycelia transition. As we further explore the structure of the synthase and other molecules in the pathway, we can further elucidate such mechanisms. <scene name='Karen_Bulaklak/Sandbox1_Glucosamine_6_Phosphate_Synthase/Serine/1'>Serine</scene> <scene name='Karen_Bulaklak/Sandbox1_Glucosamine_6_Phosphate_Synthase/Secondary_structures/1'>Secondary structures</scene>
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A physical model of the isomerase domain, the main ligand binding structure of the protein, can aid in the understanding of its multiple substrate interactions and necessary conformations for enzymatic activity.
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Future projects may include creating physical models of possible drug that target binding domains or other areas of glucosamine-6-phosphate, highlighting catalytic sites and modeling the prokaryotic form of the protein to compare their activity. With this new tool, we can propose ways to inhibit substrate binding, and consequently, the metabolic pathway of C. Albicans.
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<scene name='Karen_Bulaklak/Sandbox1_Glucosamine_6_Phosphate_Synthase/Serine/1'>Serine</scene> <scene name='Karen_Bulaklak/Sandbox1_Glucosamine_6_Phosphate_Synthase/Secondary_structures/1'>Secondary structures</scene>
<applet load='2put' size='300' frame='true' align='right' caption='Insert caption here' />
<applet load='2put' size='300' frame='true' align='right' caption='Insert caption here' />

Revision as of 03:06, 8 December 2009

Discussion

A 3-dimensional model of Glucosamine-6-phosphate synthase was created highlighting vital features of the protein’s isomerase domain:

  • The UDP-GlcNAc binding pocket, colored in dark blue, includes the sidechains Gly474, Val476, Ser484, Thr487, His492 and residues 489-491.
    • Coordinates with metal cation (in red) when substrate (in magenta) is bound, possibly indicating that a positive charge is needed to increase the stability of the enzyme-substrate complex.
  • Fructose-6-phosphate, colored in green, interacts with sidechains of Glu591, Lys588, His607 (shown in light blue). These residues form a small binding domain for F6P, which has yet to be completely characterized.
  • These two important binding sites appear on opposite sides of each chain, perhaps indicating that the substrates do not greatly effect each others’ interaction with the protein.
  • Tetramerization sites create a horizontal line of symmetry across the protein, separating identical subunits (A and D, B and C), a so-called “dimer of dimers.”
  • Beta sheets are also shown in light pink, which could provide internal support to the protein.

A physical model of the isomerase domain, the main ligand binding structure of the protein, can aid in the understanding of its multiple substrate interactions and necessary conformations for enzymatic activity.

Future projects may include creating physical models of possible drug that target binding domains or other areas of glucosamine-6-phosphate, highlighting catalytic sites and modeling the prokaryotic form of the protein to compare their activity. With this new tool, we can propose ways to inhibit substrate binding, and consequently, the metabolic pathway of C. Albicans.

Insert caption here

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Karen Bulaklak

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