Sandbox Reserved 1058: Difference between revisions

Dimethylarginine Dimethyaminohydrolase <span class="plainlinks">[http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/5/3/18.html EC 3.5.3.18]</span> (commonly known as DDAH) is a member of the <span class="plainlinks">[https://en.wikipedia.org/wiki/Hydrolase hydrolase]</span> family of enzymes which use water to break down molecules <ref name="palm">Palm F, Onozato ML, Luo Z, Wilcox CS. Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. American Journal of Physiology. 2007 Dec 1;293(6):3227-3245. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/17933965 17933965]</span> doi:<span class="plainlinks">[http://ajpheart.physiology.org/content/293/6/H3227 10.1152/ajpheart.00998.2007]</span></ref>. Additionally, DDAH is a <span class="plainlinks">[https://en.wikipedia.org/wiki/Nitric_oxide_synthase nitric oxide synthase (NOS)]</span> regulator. It metabolizes free arginine derivatives, namely <span class="plainlinks">[https://en.wikipedia.org/wiki/Asymmetric_dimethylarginine N^Ѡ,N^Ѡ-dimethyl-L-arginine (ADMA)]</span> and <span class="plainlinks">[https://en.wikipedia.org/wiki/Methylarginine N^Ѡ-methyl-L-arginine (MMA)]</span> which competitively inhibit NOS <ref name="tran">Tran CTL, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atherosclerosis Supplements. 2003 Dec;4(4):33-40. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/14664901 14664901]</span> doi:<span class="plainlinks">[http://www.sciencedirect.com/science/article/pii/S1567568803000321 10.1016/S1567-5688(03)00032-1]</span></ref>. DDAH converts MMA or ADMA to two products: <span class="plainlinks">[https://en.wikipedia.org/wiki/Citrulline L-citrulline]</span> and an amine <ref name="frey">Frey D, Braun O, Briand C, Vasak M, Grutter MG. Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: a basis for the design of specific inhibitors. Structure. 2006 May;14(5):901-911. PMID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pubmed/16698551 16698551]</span> doi:<span class="plainlinks">[http://www.sciencedirect.com/science/article/pii/S0969212606001717 10.1016/j.str.2006.03.006]</span></ref> (Figure 1). DDAH is expressed in the cytosol of cells in humans, mice, rates, sheep, cattle, and bacteria <ref name="palm" />. DDAH activity has been localized mainly to the brain, kidney, pancreas, and liver in these organisms. Presented in this page is information from DDAH isoform 1 (DDAH-1); however, there are two different isoforms <ref name="frey" />.

DDAH’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 propeller contains five repeats of a ββαβ motif <ref name="frey" />, which constitutes DDAH's overall structure as a <span class="plainlinks">[https://en.wikipedia.org/wiki/Beta-propeller beta-propeller]</span>. These motifs in DDAH form a <scene name='75/752351/Ddah_water_pore/9'>channel</scene> filled with water molecules (red spheres). Lys174 and Glu77 form a <scene name='75/752351/Ddah_salt_bridge/4'>salt bridge</scene> in the channel that forms the bottom of the <scene name='75/752351/Ddah_active_site/1'>active site</scene> for the protein. One side of the channel is a <scene name='75/752351/Ddah_water_pore/3'>water-filled pore</scene>, whereas the other side is the active site cleft that is defined by a short loop region and alpha helical structures <ref name="frey" />.

Amino acids 25-36 of DDAH constitute the loop region of the protein which is more commonly known as the lid region <ref name="frey" />. This lid region is very flexible. The lid is what allows the active site to be exposed to substrate binding or not. Studies have shown crystal structures of the lid at <scene name='69/694225/Low_ph_w_lid/1'>open</scene> and <scene name='69/694225/Closed_lid_zn9/1'>closed </scene> conformations. In the open conformation, the lid forms an alpha helix and the amino acid <scene name='69/694225/Low_ph_w_lid/2'>Leu29</scene> is moved so it does not interact with the active site. This allows the active site to be vulnerable to attack. When the lid is closed, a specific <scene name=’75/752351/Hbond_leu29/1’>hydrogen bond</scene> can form between the Leu29 carbonyl and the amino group on bound molecule. This stabilizes this complex. The Leu29 is then <scene name='69/694225/Closed_lid_zn9/3'>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" />.

Active sites of DDAH from different organisms are similar. Amino acids involved in the chemical mechanism of creating products are also <scene name='69/694225/Evolutionary_conservation/1'>conserved</scene> (Figure 4).

It was found that Cys273, His172, Glu77, Asp78, and Asp 268 all play a role in the binding of Zn(II). <scene name='69/694225/Active_site6/1'>Cys273</scene> directly coordinates with the Zn(II) ion in the active site while the other significant residues stabilize the ion via hydrogen bonding interactions with water molecules in the active site. Depending on pH, His172 can <scene name='69/694225/Active_site_9/1'>change conformation</scene> and use the <span class="plainlinks">[https://en.wikipedia.org/wiki/Imidazole imidazole]</span> group to directly coordinate the Zn(II) ion. Cys273, which is conserved between bovine and humans, is the key active site residue that coordinates Zn(II) <ref name="frey" />. Zinc-cysteine complexes have been found to be important mediators of protein <span class="plainlinks">[https://en.wikipedia.org/wiki/Catalysis catalysis]</span>, regulation, and structure <ref name="pace">Pace NJ, Weerpana E. Zinc-binding cysteines: diverse functions and structural motifs. Biomolecules. 2014 June;4(2):419-434. PMCID:<span class="plainlinks">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4101490/ 4101490]</span> doi:<span class="plainlinks">[http://www.mdpi.com/2218-273X/4/2/419/htm 10.3390/biom4020419]</span> </ref>. Cys273 and the water molecules stabilize the Zn(II) ion in a tetrahedral environment. The Zn(II) dissociation constant is 4.2 nM which is consistent with the nanomolar concentrations of Zn(II) in the cells, which provides more evidence for the regulatory use of Zn(II) by DDAH <ref name="pace" />.

<scene name='75/752351/Ddah_l-homocysteine/2'>L-homocysteine</scene> and <scene name='75/752351/Ddah_with_L-citrulline/4'>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 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. Both molecules enter the active site and cause DDAH to be in its closed lid formation. The α carbon on either molecule creates three salt bridges with DDAH: two with the guanidine group of Arg144 and one with the guanidine group Arg97. Another salt bridge is formed between the ligand and Asp72. The <scene name=’75/752351/Hbond_leu29/2’>molecules are stabilized</scene> in the active site by hydrogen bonds: 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.