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==Different Isoforms== | ==Different Isoforms== | ||
DDAH has two main isoforms <ref name="frey" />. DDAH-1 colocalizes with nNOS (neuronal NOS). This enzyme is found mainly in the brain and kidneys of organisms <ref name="tran" />. DDAH-2 is found in tissues with eNOS (endothelial NOS) <ref name="frey" />. DDAH-2 localization has been found in the heart, kidney, and placenta <ref name="tran" />. Additionally, studies show that DDAH-2 is expressed in iNOS containing immune tissues (inducible NOS) <ref name="frey" />. Both of the isoforms have conserved residues that are involved in the catalytic mechanism of DDAH (Cys, Asp, and His). The differences between the isoforms is in the substrate binding residues and the lid region residues. DDAH-1 has a positively charged lid region while DDAH-2 has a negatively charged lid. In total, three salt bridge differ between DDAH-1 and DDAH-2 isoforms <ref name="frey" />. | DDAH has two main isoforms <ref name="frey" />. DDAH-1 colocalizes with <span class="plainlinks">[https://en.wikipedia.org/wiki/Nitric_oxide_synthase nNOS (neuronal NOS)]</span>. This enzyme is found mainly in the brain and kidneys of organisms <ref name="tran" />. DDAH-2 is found in tissues with <span class="plainlinks">[https://en.wikipedia.org/wiki/Nitric_oxide_synthase eNOS (endothelial NOS)]</span> <ref name="frey" />. DDAH-2 localization has been found in the heart, kidney, and placenta <ref name="tran" />. Additionally, studies show that DDAH-2 is expressed in iNOS containing immune tissues <span class="plainlinks">[https://en.wikipedia.org/wiki/Nitric_oxide_synthase (inducible NOS)]</span> <ref name="frey" />. Both of the isoforms have conserved residues that are involved in the catalytic mechanism of DDAH (Cys, Asp, and His). The differences between the isoforms is in the substrate binding residues and the lid region residues. DDAH-1 has a positively charged lid region while DDAH-2 has a negatively charged lid. In total, three salt bridge differ between DDAH-1 and DDAH-2 isoforms <ref name="frey" />. | ||
==General Structure== | ==General Structure== | ||
DDAH-1’s <scene name='69/694225/Secondary_structure_colored/3'>secondary structure</scene> has a <scene name='69/694225/Prop_domains/2'>propeller-like fold</scene> which is characteristic of the superfamily of <span class="plainlinks">[https://en.wikipedia.org/wiki/Arginine:glycine_amidinotransferase L-arginine/glycine amidinotransferases]</span> <ref name="humm">Humm A, Fritsche E, Mann K, Göhl M, Huber R. Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue. Biochemical Journal. 1997 March 15;322(3):771-776. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1218254/ 9148748]</span> doi:<span class="plainlinks">[http://www.biochemj.org/content/322/3/771 10.1042/bj3220771]</span></ref>. This five-stranded <span class="plainlinks">[https://en.wikipedia.org/wiki/Beta-propeller propeller]</span> contains five repeats of a ββαβ motif <ref name="frey" />. These motifs in DDAH form a <scene name='75/752351/Ddah_water_pore/12'>channel</scene> filled with water molecules (red spheres). Lys174 and Glu77 form a <scene name='75/752351/Ddah_salt_bridge/5'>salt bridge</scene> in the channel that | DDAH-1’s <scene name='69/694225/Secondary_structure_colored/3'>secondary structure</scene> has a <scene name='69/694225/Prop_domains/2'>propeller-like fold</scene> which is characteristic of the superfamily of <span class="plainlinks">[https://en.wikipedia.org/wiki/Arginine:glycine_amidinotransferase L-arginine/glycine amidinotransferases]</span> <ref name="humm">Humm A, Fritsche E, Mann K, Göhl M, Huber R. Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue. Biochemical Journal. 1997 March 15;322(3):771-776. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1218254/ 9148748]</span> doi:<span class="plainlinks">[http://www.biochemj.org/content/322/3/771 10.1042/bj3220771]</span></ref>. This five-stranded <span class="plainlinks">[https://en.wikipedia.org/wiki/Beta-propeller propeller]</span> contains five repeats of a ββαβ motif <ref name="frey" />. These motifs in DDAH form a <scene name='75/752351/Ddah_water_pore/12'>channel</scene> filled with water molecules (red spheres). Lys174 and Glu77 form a <scene name='75/752351/Ddah_salt_bridge/5'>salt bridge</scene> in the channel that makes up the bottom of the <scene name='75/752351/Ddah_active_site/3'>active site</scene>, shown here filled with water molecules. One side of the channel is a <scene name='75/752351/Ddah_water_pore/13'>water-filled pore</scene>, whereas the other side is the active site cleft <ref name="frey" />. | ||
===Lid Region=== | ===Lid Region=== | ||
Amino acids 25-36 of DDAH constitute the flexible | Amino acids 25-36 of DDAH constitute the flexible | ||
<scene name='75/752351/Lid_focus/2'>loop region</scene> of the protein, which is more commonly known as the lid region <ref name="frey" /> | <scene name='75/752351/Lid_focus/2'>loop region</scene> of the protein, which is more commonly known as the lid region <ref name="frey" />. Studies have shown crystal structures of the lid at <scene name='69/694225/Open_surface/8'>open</scene> and <scene name='69/694225/Closed_surface/5'>closed</scene> conformations. In the open conformation, the lid forms an <scene name='69/694225/Lid_helix/2'>alpha helix</scene> and the amino acid Leu29 is moved so it does not interact with the active site, thus allowing the active site to be vulnerable to attack. When the lid is closed, a <scene name='75/752351/Hbond_leu29/3'>hydrogen bond</scene> can form between the Leu29 carbonyl and the amino group on a bound molecule. This hydrogen bond stabilizes the substrate in the active site. The Leu29 is then <scene name='75/752351/Hbond_leu29/5'>blocking</scene> the active site entrance <ref name="frey" />. Opening and closing the lid takes place faster than the actual reaction in the active site <ref name="rasheed">Rasheed M, Richter C, Chisty LT, Kirkpatrick J, Blackledge M, Webb MR, Driscoll PC. Ligand-dependent dynamics of the active site lid in bacterial Dimethyarginine Dimethylaminohydrolase. Biochemistry. 2014 Feb 18;53:1092-1104. PMCID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945819/ PMC3945819]</span> doi:<span class="plainlinks">[http://pubs.acs.org/doi/abs/10.1021/bi4015924 10.1021/bi4015924]</span></ref>. This suggests that the <span class="plainlinks">[https://en.wikipedia.org/wiki/Rate-determining_step rate-limiting step]</span> of this reaction is not the lid movement, but is the actual chemistry happening to the substrate in the active site of DDAH <ref name="rasheed" />. | ||
====Lid Region Conservation==== | ====Lid Region Conservation==== | ||
The specific residues in the lid region | The specific residues in the lid region vary between organisms <ref name="frey" /> (Figure 2). The only consistent similarity is a <span class="plainlinks">[https://en.wikipedia.org/wiki/Conserved_sequence conserved]</span> leucine residue in this lid that functions to hydrogen bond with the <span class="plainlinks">[https://en.wikipedia.org/wiki/Ligand ligand]</span> bound to the active site in DDAH-1 but not in DDAH-2 <ref name="rasheed" /> (Figure 2). Different <span class="plainlinks">[https://en.wikipedia.org/wiki/Protein_isoform isoforms]</span> from the same species can have differences in lid regions as well <ref name="frey" />. DDAH-2 has a negatively charged lid while DDAH-1 has a positively charged lid <ref name="frey" />. | ||
[[Image:Lid Region WebLogo.png|500 px|center|thumb|'''Figure 2.''' WebLogo for the lid region in DDAH-1 of eight different organisms.]] | [[Image:Lid Region WebLogo.png|500 px|center|thumb|'''Figure 2.''' WebLogo for the lid region in DDAH-1 of eight different organisms.]] | ||
| Line 25: | Line 25: | ||
====Channel with Salt Bridge and Water Pore==== | ====Channel with Salt Bridge and Water Pore==== | ||
There is a channel in the center of the protein that is closed by a <scene name='75/752351/Ddah_salt_bridge/6'>salt bridge</scene> connecting Glu77 and Lys174 <ref name="frey" />. This salt bridge constitutes the bottom of the active site. There is a pore containing water on one side of the channel. This pore is <scene name='75/752351/Ddah_water_pore/14'>delineated</scene> by the first β strand of each of the five propeller blades. The water in the | There is a channel in the center of the protein that is closed by a <scene name='75/752351/Ddah_salt_bridge/6'>salt bridge</scene> connecting Glu77 and Lys174 <ref name="frey" />. This salt bridge constitutes the bottom of the active site. There is a pore containing water on one side of the channel. This pore is <scene name='75/752351/Ddah_water_pore/14'>delineated</scene> by the first β strand of each of the five propeller blades. The water in the channel forms hydrogen bonds to <scene name='75/752351/Ddah_water_pore/15'>His172 and Ser175</scene>. | ||
====Active Site Conservation==== | ====Active Site Conservation==== | ||
| Line 32: | Line 32: | ||
====Zn(II) Bound to the Active Site==== | ====Zn(II) Bound to the Active Site==== | ||
<span class="plainlinks">[https://en.wikipedia.org/wiki/Zinc Zinc (Zn(II))]</span> acts as an <span class="plainlinks">[https://en.wikipedia.org/wiki/Endogeny_(biology) endogenous]</span> inhibitor of DDAH <ref name="frey" /> (Figure 5). The Zn(II)-binding site is located inside the protein’s active site, making it a <span class="plainlinks">[https://en.wikipedia.org/wiki/Competitive_inhibition competitive inhibitor]</span>. When bound, Zn(II) <scene name='69/694225/Closed_lid_zn9/6'>blocks the entrance</scene> of any other substrate. When <span class="plainlinks">[https://en.wikipedia.org/wiki/Crystallization crystallized]</span> with Zn(II) at <scene name='69/694225/Active_site6/2'>pH 6.3</scene>, an open conformation of the lid region has been shown; however, when Zn(II) is bound at <scene name='69/694225/Active_site_9/3'>pH 9.0</scene>, a closed lid has been observed (Figure 5). | |||
[[Image:Zn(II) bound at differing pH values.jpg|500 px|center|thumb|'''Figure 5.''' Zn(II) bound to the active site of DDAH at differing pH values. A) Zn(II) bound at pH 9.0 showing the channel of DDAH. B) Zn(II) bound at 9.0 showing the closed conformation lid with Leu29 blocking the active site. C) Zn(II) bound at pH 6.3 showing the channel of DDAH. D) Zn(II) bound at pH 6.3 showing the open lid conformation with Leu29 away from the active site.]] | [[Image:Zn(II) bound at differing pH values.jpg|500 px|center|thumb|'''Figure 5.''' Zn(II) bound to the active site of DDAH at differing pH values. A) Zn(II) bound at pH 9.0 showing the channel of DDAH. B) Zn(II) bound at 9.0 showing the closed conformation lid with Leu29 blocking the active site. C) Zn(II) bound at pH 6.3 showing the channel of DDAH. D) Zn(II) bound at pH 6.3 showing the open lid conformation with Leu29 away from the active site.]] | ||
=====Important residues in Zinc Binding===== | =====Important residues in Zinc Binding===== | ||
| Line 39: | Line 39: | ||
====Inhibitors==== | ====Inhibitors==== | ||
<scene name='75/752351/Ddah_l-homocysteine/3'>L-homocysteine</scene> and <scene name='75/752351/Ddah_with_l-citrulline/5'>L-citrulline</scene> bind in the active site in the same orientation as MMA and ADMA to create the same <span class="plainlinks">[https://en.wikipedia.org/wiki/Intermolecular_force intermolecular bonds]</span> between them and DDAH <ref name="frey" /> (Figure 6). L-citrulline is also a product of DDAH hydrolyzing ADMA and MMA, suggesting DDAH activity creates a <span class="plainlinks">[https://en.wikipedia.org/wiki/Negative_feedback negative feedback]</span> loop on itself (Figure 3). Both molecules enter the active site and cause DDAH to be in its closed lid formation. The α carbon on either molecule creates three <scene name='75/752351/Hbond_leu29/7'>salt bridges</scene> | <scene name='75/752351/Ddah_l-homocysteine/3'>L-homocysteine</scene> and <scene name='75/752351/Ddah_with_l-citrulline/5'>L-citrulline</scene> bind in the active site in the same orientation as MMA and ADMA to create the same <span class="plainlinks">[https://en.wikipedia.org/wiki/Intermolecular_force intermolecular bonds]</span> between them and DDAH <ref name="frey" /> (Figure 6). L-citrulline is also a product of DDAH hydrolyzing ADMA and MMA, suggesting DDAH activity creates a <span class="plainlinks">[https://en.wikipedia.org/wiki/Negative_feedback negative feedback]</span> loop on itself (Figure 3). Both molecules enter the active site and cause DDAH to be in its closed lid formation. The α carbon on either molecule creates three <scene name='75/752351/Hbond_leu29/7'>salt bridges</scene> with DDAH: two with the guanidine group of Arg144 and one with the guanidine group on Arg97. Another salt bridge is formed between the ligand and Asp72. The molecules are stabilized in the active site by <scene name='75/752351/Hbond_leu29/4'>four hydrogen bonds</scene>: α carbon-amino group of the ligand to main chain carbonyls of Val267 and Leu29. Hydrogen bonds also form between the side chains of Asp78 and Glu77 with the ureido group of L-citrulline. | ||
Like L-homocysteine and L-citrulline, <scene name='75/752351/Ddah_s-nitroso-l-homocysteine/4'>S-nitroso-L-homocysteine</scene> binds and the lid region of DDAH is closed (Figure 6). When DDAH reacts with S-nitroso-L-homocysteine, a covalent product, N-thiosulfximide exist in the active site because of its binding to Cys273. N-thiosulfximide is stabilized by several salt bridges and hydrogen bonds. Arg144 and Arg97 stabilize the α carbon-carbonyl group via salt bridges, and Leu29, Val267, and Asp72 stabilize the α carbon-amino group by forming hydrogen bonds <ref name="frey" />. | Like L-homocysteine and L-citrulline, <scene name='75/752351/Ddah_s-nitroso-l-homocysteine/4'>S-nitroso-L-homocysteine</scene> binds and the lid region of DDAH is closed (Figure 6). When DDAH reacts with S-nitroso-L-homocysteine, a covalent product, N-thiosulfximide exist in the active site because of its binding to Cys273. N-thiosulfximide is stabilized by several salt bridges and hydrogen bonds. Arg144 and Arg97 stabilize the α carbon-carbonyl group via salt bridges, and Leu29, Val267, and Asp72 stabilize the α carbon-amino group by forming hydrogen bonds <ref name="frey" />. | ||
[[Image:L-citrulline, L-homocysteine, and S-nitroso-L-homocysteine.jpg|500px|center|thumb|'''Figure 6.''' Structures of DDAH inhibitors.]] | [[Image:L-citrulline, L-homocysteine, and S-nitroso-L-homocysteine.jpg|500px|center|thumb|'''Figure 6.''' Structures of DDAH inhibitors.]] | ||
==Clinical Relevance== | ==Clinical Relevance== | ||
DDAH works to hydrolyze MMA and ADMA <ref name="frey" />. Both MMA and ADMA competitively inhibit NO synthesis by inhibiting Nitric Oxide Synthase (NOS). NO is made by NOS creating L-citrulline from <span class="plainlinks">[https://en.wikipedia.org/wiki/Arginine L-arginine]</span> <ref name="frey" />. If DDAH is overexpressed, NOS | DDAH works to hydrolyze MMA and ADMA <ref name="frey" />. Both MMA and ADMA competitively inhibit NO synthesis by inhibiting Nitric Oxide Synthase (NOS). NO is made by NOS creating L-citrulline from <span class="plainlinks">[https://en.wikipedia.org/wiki/Arginine L-arginine]</span> <ref name="frey" />. If DDAH is overexpressed, NOS activity will subsequently increase <ref name="frey" />. ADMA and MMA can <span class="plainlinks">[https://en.wikipedia.org/wiki/Enzyme_inhibitor inhibit]</span> the synthesis of NO by competitively inhibiting all three kinds of NOS (endothelial, neuronal, and inducible) <ref name="frey" />. Underexpression or inhibition of DDAH decreases NOS activity and NO levels will decrease. Because of <span class="plainlinks">[https://en.wikipedia.org/wiki/Nitric_oxide nitric oxide’s (NO)]</span> role in signaling and defense, NO levels in an organism must be regulated to reduce damage to cells <ref name="janssen">Janssen W, Pullamsetti SS, Cooke J, Weissmann N, Guenther A, Schermuly RT. The role of dimethylarginine dimethylaminohydrolase (DDAH) in pulmonary fibrosis. The Journal of Pathology. 2012 Dec 12;229(2):242-249. Epub 2013 Jan. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/23097221 23097221]</span> doi:<span class="plainlinks">[http://onlinelibrary.wiley.com/doi/10.1002/path.4127/references;jsessionid=C34C6C633A21C2ECE14278BBC902AD71.f03t04?globalMessage=0 10.1002/path.4127]</span></ref>. NO is an important signaling and effector molecule in <span class="plainlinks">[https://en.wikipedia.org/wiki/Neurotransmission neurotransmission]</span>, bacterial defense, and regulation of vascular tone <ref name="colasanti">Colasanti M, Suzuki H. The dual personality of NO. ScienceDirect. 2000 Jul 1;21(7):249-252. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/10979862 10979862]</span> doi:<span class="plainlinks">[http://www.sciencedirect.com/science/article/pii/S0165614700014991 10.1016/S0165-6147(00)01499-1]</span></ref>. Because NO is highly toxic, freely diffusible across membranes, and its radical form is fairly reactive, cells must maintain a large control on concentrations by regulating NOS activity and the activity of enzymes such as DDAH that have an indirect effect of the concentration of NO <ref name="rassaf">Rassaf T, Feelisch M, Kelm M. Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues. Free Rad. Biol. Med. 2004 Feb 15;36(4):413-422. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/14975444 14975444]</span> doi:<span class="plainlinks">[http://www.sciencedirect.com/science/article/pii/S0891584903007962 10.1016/j.freeradbiomed.2003.11.011]</span></ref>. An imbalance of NO contributes to several diseases. Low NO levels, potentially caused by low DDAH activity and therefore high MMA and ADMA concentrations, have been associated with diseases such as <span class="plainlinks">[https://en.wikipedia.org/wiki/Uremia uremia]</span>, <span class="plainlinks">[http://www.mayoclinic.org/diseases-conditions/heart-failure/basics/definition/con-20029801 chronic heart failure]</span>, <span class="plainlinks">[https://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis]</span>, and <span class="plainlinks">[https://en.wikipedia.org/wiki/Hyperhomocysteinemia hyperhomocysteinemia]</span> <ref name="tsao">Tsao PS, Cooke JP. Endothelial alterations in hypercholesterolemia: more than simply vasodilator dysfunction. Journal of Cardiovascular Pharmacology. 1998;32(3):48-53. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/9883748 9883748]</span></ref>. High levels of NO have been involved with diseases such as <span class="plainlinks">[https://en.wikipedia.org/wiki/Septic_shock septic shock]</span>, <span class="plainlinks">[http://www.mayoclinic.org/diseases-conditions/migraine-headache/home/ovc-20202432 migraine]</span>, <span class="plainlinks">[https://en.wikipedia.org/wiki/Inflammation inflammation]</span>, and <span class="plainlinks">[https://en.wikipedia.org/wiki/Neurodegeneration neurodegenerative disorders]</span> <ref name="vallance">Vallance P, Leiper J. Blocking NO synthesis: how, where and why? Nat. Rev. Drug Discov. 2002 Dec;1(12):939-950. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/12461516 12461516]</span> doi:<span class="plainlinks">[http://www.nature.com/nrd/journal/v1/n12/full/nrd960.html 10.1038/nrd960]</span></ref>. Because of the effects on NO levels and known inhibitors to DDAH, regulation of DDAH may be an effective way to regulate NO levels, therefore treating these diseases <ref name="frey" />. Additionally, researchers can take advantage of the fact that there are two different isoforms of this enzyme and create drugs that target one isoform over another to control NO levels in specific tissues in the body <ref name="frey" />. | ||
</StructureSection> | </StructureSection> | ||