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=='''Leadzyme, 1nuv'''==
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=='''Pantetheinase (4CYG)<ref>PMID: 25478849 </ref>'''==
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<Structure load='1nuv' size='500' frame='true' align='right' caption='Leadzyme 1nuv' scene='Insert optional scene name here' />
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by [Luke Schnitzler, Patrick Tonne, Owen O'Connor, Tyler Russell, Nicholas Sant]
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===Introduction===
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[[Student Projects for UMass Chemistry 423 Spring 2016]]
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Leadzyme is a ribozyme, created ''in vitro'' through artificial selection. In the presence of lead, leadzyme will bind and cut RNA. The cleavage reaction is a two step process. First the ribozyme uses the bound lead ion to cleave the phosphodiester backbone of the substrate strand. This produces both a free 5' hydroxyl group and a 2',3' cyclic phosphodiester. The cyclic phosphate is then hydrolyzed yielding a 3'-phosphate. Leadzyme has two different states which it transitions between.
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<StructureSection load='4CYG' size='350' side='right' caption='caption for Molecular Playground (PDB entry [[4CYG]])' scene=''>
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<br> The first is the <scene name='Sandbox_Reserved_434/Pre_catalytic_interaction/2'>pre-catalytic state</scene>. This state happens when lead binds causing <font color='yellow'>C23</font> to align with the scissile <font color='teal'>G24</font> phosphodiester bond. In the ground state there is no ion pulling these residues so they are farther apart. <font color='orange'>It is believed that <scene name='Sandbox_Reserved_434/Sr_binding_site/5'>strontium ions</scene> may act as a competitive inhibitors for this ribozyme.</font> <font color='purple'>Strontium is hydrated and binds to ribonucleotides 23,26 and 43-45.</font> The interactions formed are hydrogen bonds via the water and the bases. The strontium binding sites overlap with the lead binding sites, leading to the belief that it may act as a competitive inhibitor.<font color='green'> <scene name='Sandbox_Reserved_434/Mg_binding_site/3'>Magnesium</scene> is also a known inhibitor of this ribozyme, but it is believed to be an allosteric inhibitor.</font> <font color='blue'>It has the different binding sites across bases 19-20,24-25,29-30,39-43,47.</font> When it binds it causes conformational changes within the ribozyme that disrupt its normal cleavage functions and return it to its ground state conformation. <scene name='Sandbox_Reserved_434/Allosteric_effect_of_mg/1'>Magnesium forms a bridge</scene> between<font color='red'> G42 and G43</font> and the substrate strand of RNA which constrains the trinucleotide bulge. Without this interaction, the ribozyme relaxes into its precatalytic state.
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<br> Leadzyme is thought to have similarities with 5s subunit of the rRNA of ribosomes. This has lead people to the hypothesis that lead poisoning stems from ribosomal malfunction due to the presence of lead ions. There are D-loops of the rRNA have similar sequences and when exposed to lead can have cleavage activity. If the concentration of lead in the body is enough to interact with a significant number of ribosomes they could begin to cleave necessary mRNAs, which would be detrimental to the body.
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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===Overall Structure===
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==Introduction==
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<Structure load='1nuv' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
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Vanin 1, otherwise known as pantetheinase, is an enzyme found throughout the body in various tissues including the liver and kidneys. As an ectoenzyme—any enzyme found on the outside or outer surface of a cell—pantethiense is anchored to the cell wall by a glycosylphosphatidylinositol (GPI) linker, allowing for the it to carry out its enzymatic purpose of hydrolyzing pantetheine to pantothenic acid and cysteamine [1]. The two protein subunits possess dense regions of <scene name='48/483891/Secondary_structure/1'>beta strands and alpha helices</scene> which create binding sites for three types of ligands, the non-polar <scene name='48/483891/Rrv/1'>RRV ligand</scene>, a di(hydroxyethyl)ether compound <scene name='48/483891/Peg/1'>PEG</scene>, and <scene name='48/483891/Nag/1'>NAG</scene> (N-acetyl-d-glucosamine) ligands. The identification of the associated binding sites has led to the investigation of active-site inhibitors (see Additional Features).
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Leadzyme is made of a RNA duplexe shown here <scene name='Sandbox_Reserved_434/Rna_duplex_subunit/2'>here</scene> with nucleotides 18-30 shown in orange and nucleotides 39-49 shown in blue. The duplex contains two <scene name='Sandbox_Reserved_434/Non_watson-crick_base_pairs/1'>Non Watson-Crick Base Pairs.</scene> The C23 to A45 is shown in black and A25 to G44 is shown in blue, which form a 3 nucleotide bulge. Divalent ions bridge the complementary strands by binding tandem purines from one strand to the major groove. The scissile phosphodiester bond fits at at the junction between the duplex and the bulge. The nucleotides forming the bulge have been shown to be relatively mobile and 2 significantly different confirmations have been documented. The main difference between the confirmations is that in one the all nucleotides in the bulge point away from the interior of the duplex while in the other confirmation the A in the bulge points inward.
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===Binding Interactions===
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The importance of vanin 1 lies in the products of the enzymatic reaction. Pantothenic acid (vitamin B12) plays a significant role in the maintenance of the nervous system and brain. The compound is also involved in DNA synthesis, as well as fatty acid and amino acid metabolism [2]. Cysteamine—a product of the degradation of the amino acid cysteine—is used to form coenzyme A, a compound that plays a key role in the citric acid cycle and the synthesis of fatty acids. A lack of these biomolecules can lead to significant physiological issues (see Additional Features).
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<Structure load='1nuv' size='500' frame='true' align='right' caption='Special features of leadzyme.' scene='Insert optional scene name here' />
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Pb2+ in leadzyme cleaves the RNA by cleaving the substrate’s backbone and producing a 2’,3’-cyclic phosphodiester and a 5’-hydroxyl. This first step is common among many ribozymes, however, the 2’,3’-cyclic phosphodiester is hydrolyzed to produce a 3’-phosphodiester.
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Leadzyme requires Pb2+ to cleave RNA and is inhibited by Mg2+, through competitive inhibition. A possible suggestion for Mg2+ inhibition may have to do with the Mg(H2O)6 which bind in the major groove at the G39, G30 and A29 nucleotides at one location (<scene name='Sandbox_Reserved_434/Site_i/2'>Site I</scene>, in both the ground state and pre-catalytic state), at the U47, G20, and G19 nucleotides from another location (<scene name='Sandbox_Reserved_434/Site_iii/3'>Site III</scene>, from both states), and at the U41, G42, G43, G24, and A25 nucleotides in the ground state (<scene name='Sandbox_Reserved_434/U41_g42_g43_g24_and_a25/3'>Site II</scene>). This last interaction is particularly important since it “ligates” the <scene name='Sandbox_Reserved_434/Trinucleotide_bulge/4'>Trinucleotide Bulge</scene>, which inhibits the activity of the leadzyme. Since this type of interaction occurs only in the ground state, it is thought to stabilize the ground state and thus prevent catalysis of RNA cleavage. The Mg2+ "pulls in" the trinucleotide bulge by hydrogen bonding with<font color = 'red'>N7 and O6 in G42 and O4 in U41</font> with a "sphere" of six water molecules attached to the magnesium ion. It is worth noting that because of the competition between metal ions in leadzyme, if lead is added to the ground state form of leadzyme with bound Mg2+ at site II, the structure may change from ground state to pre-catalytic by interrupting the hydrogen bonding/bridging between the two tandem purines (G42 & G43) and A25 and G24, thus relaxing the trinucleotide bulge.There are some differences in the types of binding involved in sites II than in sites I & III. For example. In site II, O6 at the G43 nucleotide shares an H2O with O6 of G42 rather than hydrogen bonding with a separate water molecule.
 
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Another ion of interest is Sr2+, which binds similarly as Pb2+ but has different catalytic effects due to electronic differences. Similar bonding occurs because of similar ionic radii (Sr2+=1.12A; Pb2+=1.13A). Although there are multiple major Sr2+ binding sites, one particularly important site in the pre-catalytic form interacts with <scene name='Sandbox_Reserved_434/Sr_site_iii/2'>G24, G23, and A45</scene>, which is located in the Scissile Bond region. At this bonding site, Sr2+ ligates N1 of A45 and non bridging phosphate of A25. This bonding dramatically changes the conformation of the phosphodiester backbone (in the presence of Mg2+ and Sr2). In the ground state <scene name='Sandbox_Reserved_434/Ground_state_sr/4'>Sr2+ binds to G43, G44, and A45</scene>, which helps stabilize the ground state. As with Mg2+, there is a H2O sphere around Sr2+ and this allows hydrogen bonding between <font color = 'darkorange'>N7 of G23, N6 of A 45, O6 of G44</font>, and a "nonbriding phosphate oxygen of G43". Also, it is important to notice that the binding of Sr2+ is adjacent to Site II of Mg2+ in the ground state and has very little affect on the binding of Mg2+ at this site. It is important that metal ions such as Sr2+ and Mg2+ inhibit leadzyme since without this "stable" crystal structure, leadzyme would continuously cleave RNA, including itself.
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==Overall Structure==
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The protein 4CYG has a two chain structure. The chains have identical sequences, consisting of 506 total residues each. There are 87 residues missing from the model, which have been replace with spherical basket-like structures. The structure of only Chain A has been isolated <scene name='48/483891/Chain_a_rainbow/4'>here</scene> in order to provide a more simple view. Chain A is colored to show the sequence direction <scene name='48/483891/Chain_a_rainbow/5'>here</scene>
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Because of such interactions, leadzyme in the presence of only Sr2+ does not show significant cleavage, where leadzyme in the presence of Sr2+ and Pb2+ shows reduced cleavage in comparison to leadzyme and only Pb2+.
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The secondary structure of 4CYG is made up of both beta sheets and alpha helices. The alpha helices of one chain, of which there are 11, are isolated <scene name='48/483891/Alphas_only/2'>here</scene>, where the polar side of the helix is pink and the hydrophobic side is gray. The beta sheets of one chain, colored by polarity, can be viewed <scene name='48/483891/Betas_only_polar/1'>here</scene>. The polar portions are colored pink and nonpolar portions are colored grey, allowing for a visual representation of the alternating sequence.
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===Additional Features===
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4CYG has three types ligands involved in the structure. There are two <scene name='48/483891/Rrv/1'>RRV</scene> ((2r)-2,4-dihydroxy-n-[(3s)-3-hydroxy-4-phenylbutyl]-3,3-dimethylbutanamide) ligands, two <scene name='48/483891/Peg/1'>PEG</scene> (di(hydroxyethyl) ether) ligands, and eight <scene name='48/483891/Nag/1'>NAG</scene> (N-acetyl-d-glucosamine) ligands.
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<Structure load='1nuv' size='500' frame='true' align='right' caption='Special features of leadzyme.' scene='Insert optional scene name here' />
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==Binding Interactions==
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The <scene name='Sandbox_Reserved_434/Tf_all/2'>leadzyme</scene> is dependent on the inclusion of lead for catalytic effect: no other ion gives the same result. The particular area of interest for the cleavage activity occurs at a <scene name='Sandbox_Reserved_434/Tf_all/3'>bulge loop</scene>.
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4CYG is a key protein that is involved in the breakdown of pantetheine to panthothenic acid and cysteamine. These proteins are associated with many metabolic diseases like type 2 diabetes. Understanding the binding interaction would give insight into treating these diseases more effectively. 4CYG has three <scene name='48/483891/Binding_site/1'>Catalytic Residues</scene> (Glu79, Lys178 and Cys211) that represents the active site of the enzyme. The purple amino acids represent the three amino acids directly involved in the binding interactions. The active site is located in the center of the enzyme in between the two sub-units. It was discovered that Glu79 and Lys178 were responsible for orienting and activating Cys211 to catalyze the reaction. The substrate forms a covalent bond with Cys211 producing the transition state.[1]
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The catalytic cleavage depends on the presence of lead (II) in solution, and is further catalyzed by the addition of a second metal ion, particularly magnesium (II) or lanthanide ions. Strontium (II) appears to occupy the same positions as lead (II), and is used for imaging purposes, but does not have any catalytic action.
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In addition, the two residues <scene name='48/483891/Glu249_and_glu439/2'>GLU249 AND GLU439</scene> are essential for enzymatic function too. The purple regions are polar whereas the grey regions are hydrophobic. The two black amino acids represent GLU249 and GLU439. The two glutamic acid residues are both located in hydrophobic regions 4 Angstroms away. Although this is energetically unfavorable to have a polar amino acid in a nonpolar region, these two amino acids help maintain the structure between the two sub-units for proper binding interactions to occur.[1]
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Studies show that equimolar solutions of neodymium (III) and lead (II) maximize this effect, suggesting that neodymium acts in concert with lead, as an acid catalyst. The site at which the RNA is cleaved remains unchanged.<ref> Ohmichi, T., Sugimoto, N. (1996) ''FEBS Letters 393'', 97-100. </ref> <ref> PMID: 9132001 </ref> As shown by Wedekind and McKay, the lead ion may interact with the major grove of the base pairs near or in the loop area, which the secondary ion is located a few residues to either side. Both metals hydrogen bond through their hexaaquo coordination spheres.
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==Additional Features==
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The biological importance of pantetheinase is found in the products cysteamine and vitamin B5, formed from the hydrolysis reaction shown below. Cysteamine and vitamin B5 are key components in the synthesis of other necessary bio-molecules such as acetylcholine and coenzyme A.
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The actions of both the lead ion and the secondary metal ion appears to be highly pH dependent. Though variation in the pKa values changing the protonated forms of functional groups are more frequently a concern for amino acids for which more pKa's are within the range of biological pH, the leadzyme provides an example where the rate shifts are also associated strongly with pH.
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[[Image:rxn.png]]
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===Inhibition===
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The importance of pantetheinase stems from the vitality of the components of the reaction it catalyzes. It is for this reason that pantetheinase has been a promising point of research in the field of medicine. Pantetheine analogues known as pantothenamides have been shown to act as effective antibiotics that protect the body from bacterial intruders. The similarity of these analogues to pantetheine allows for the active sites on pantetheinase to catalyze their breakdown through hydrolysis. The administration of RR6 in the presence of pantetheinase and other antibiotic pantothenamides revealed that RR6 acts as a competitive inhibitor with great affinity for the active site Cys at <scene name='48/483891/Rr6/2'>residue 211</scene> on pantetheinase, thus preserving desired concentrations of the pantothenamides with antibiotic characteristics. The RR6 inhibitor is shown below.
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The leadzyme <ref>PMID: 12911297</ref> rate demonstrates a strong dependence on pH. The pKa values for the residues at the active site loop are affected by their environment. Activity has been found to be highly dependent on pH near to the biological range, with the maximum rate for lead-only solution occuring at 7, and for lead and magnesium is solution at 7.2. The rate constant increases linearly, increasing almost 100-fold from pH of 5.5 to 7.2. <ref>PMID: 8068631</ref>
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[[Image:RR6.png]]
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===Absence===
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Vitamin B5 is the recycled product of the hydrolysis of pantetheine and is a major substrate in the formation of coenzyme A (CoA). Without pantetheinase, the hydrolysis of pantetheine would occur at a significantly slower rate and would therefore produce vitamin B5 and cysteamine at a slower rate. Lower concentrations of Vitamin B5 would result in inadequate prodution of CoA and therefore inadequate production of acetylcholine. Acetylcholine is a neurotransmitter that is needed for proper brain function which means an absence of pantetheinase could lead to neurological complications.
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Cysteamine is important in the regulation of cystine levels in lysosomes. A lack of cysteamine due to the absence of pantetheinase would lead to a build up of lysosomal cystine, a disease known as cystinosis.
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The <scene name='Sandbox_Reserved_434/Ph_1/5'>duplex</scene> shows residues with <font color='red'>pKa < 3.1</font>, <font color='light blue'>pKa of 4-5</font> and <font color=00007C >pKa > 5</font>. The pH closest to the biological range, residue A45, is an adenine that participates in coordination of the lead at one of three identified lead binding sites. This adenine is also part of the only non-Watson-Crick pair near the active site. The loop connecting the two strands consists of adenines with pKa around 3.5.<ref> Legault, P., Pardi, A. (1997) ''J. Am. Chem. Soc. 119'', 6621-6628</ref> The <scene name='Sandbox_Reserved_434/Ph_2/3'>asymmetrical bulge</scene> does not exhibit an considerable increase in stability at lower pH as experienced by some similar sequences,<ref>PMID: 19485416 </ref> which would be consistent with the pH instead changing the lead binding capability.
 
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===Credits===
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==Quiz Question 1==
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Introduction - Jeffrey Salemi
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Pantetheinase exhibits an extremely similar sequence to the other proteins in the Vanin family. In addition, <scene name='48/483891/Pantetheinase/1'>pantetheinase</scene> is also sequentially similar to this enzyme, which allows the body to utilize a certain vitamin:
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Overall Structure - Adam Ramey
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a. gastric lipase
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Binding Interactions- Nicholas Vecchiarello
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b. biotinidase
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Additional Features - Tom Foley
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c. carboxypeptidase
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===References===
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d. pepsin
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==See Also==
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*[[1h1t]]
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*[[1mop]]
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*[[1esm]]
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==Credits==
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Introduction - Patrick Tonne
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Overall Structure - Luke Schnitzler
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Drug Binding Site - Owen O'Connor
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Additional Features - Nick Saint
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Quiz Question 1 - Tyler Russell
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==References==
<references/>
<references/>
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[2] "Pantetheinase." VNN1. UniProt, n.d. Web. 10 Apr. 2016.
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[3]Jansen, Patrick, and Joost Schalkwijk. "Chemical Biology Tools to Study Pantetheinases of the Vanin Family." Biochemical Society Transactions. Portland Press, n.d. Web. 10 Apr. 2016.
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[4]"Cystinosis." Genetics Home Reference. U.S. National Library of Medicine, 8 Apr. 2016. Web. 10 Apr. 2016.

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This Sandbox is Reserved from January 19, 2016, through August 31, 2016 for use for Proteopedia Team Projects by the class Chemistry 423 Biochemistry for Chemists taught by Lynmarie K Thompson at University of Massachusetts Amherst, USA. This reservation includes Sandbox Reserved 425 through Sandbox Reserved 439.


Pantetheinase (4CYG)[1]

by [Luke Schnitzler, Patrick Tonne, Owen O'Connor, Tyler Russell, Nicholas Sant]

Student Projects for UMass Chemistry 423 Spring 2016

caption for Molecular Playground (PDB entry 4CYG)

Drag the structure with the mouse to rotate
Retrieved from "http://52.214.119.220/wiki/index.php/Sandbox_Reserved_434"
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