From Proteopedia
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| | =General Structure= | | =General Structure= |
| - | DDAH’s structure has a propeller-like fold which is characteristic of the superfamily of L-arginine/glycine amidinotransferases. This five-stranded propeller contains five repeats of a ββαβ motif. This motif consists of two beta-sheets that are anti-parallel, an alpha helix, and another beta-sheet that is parallel to the second beta-sheet in the motif. These motifs in DDAH form a channel in the center of the protein structure. Lys174 and Glu77 form a salt bridge in the channel that forms the bottom of the active site for the protein. One side of the channel is a water-filled pore, whereas the other side is the active site cleft that is defined by a short loop region and alpha helical structures.
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| | ==Lid Region== | | ==Lid Region== |
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| | ===Zn2+ Bound to the Active Site=== | | ===Zn2+ Bound to the Active Site=== |
| - | In DDAH, Zinc (Zn2+) acts as an endogenous inhibitor and prevents normal NOS activity. The Zn(II)-binding site is located inside the protein’s active site, which makes it a competitive inhibitor. When bound, Zn(II) blocks the entrance of any other substrate. It was found that Cys273, His172, Glu77, Asp78, and Asp 268 all play a role in the binding of Zn(II). Cys273 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 change conformations and use the imidazole 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). Zinc-cysteine complexes have been found to be important mediators of protein catalysis, regulation, and structure. 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, thus provides more evidence for the regulatory use of Zn(II) by DDAH.
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| | [[Image:2CI6_with_Zn2+_bound.png|100 px|left|thumb|Figure Legend]] | | [[Image:2CI6_with_Zn2+_bound.png|100 px|left|thumb|Figure Legend]] |
| | <scene name='75/752348/Lid_region_with_zinc_at_low_ph/1'></scene> | | <scene name='75/752348/Lid_region_with_zinc_at_low_ph/1'></scene> |
Revision as of 12:27, 28 March 2017
Dimethylarginine Dimethylaminohydrolase
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Introduction
Dimethylarginine Dimethyaminohydrolase (commonly known as DDAH) is a member of the hydrolase family of enzymes which use water to break down molecules (palm). Specifically, DDAH is a nitric oxide synthase (NOS) regulator. Its metabolizes free arginine derivatives, namely NѠ,NѠ-dimethyl-L-arginine (ADMA) and NѠ-methyl-L-arginine (MMA) which competitively inhibit NOS [1].
DDAH is expressed in the cytosol of cells in humans, mice, rates, sheep, cattle, and bacteria [2]. DDAH activity has been localized mainly to the brain, kidney, pancreas, and liver in these organisms. If DDAH is overexpressed, NOS can be activated [3]. ADMA and MMA can inhibit the synthesis of NO by competitively inhibiting all three kinds of NOS (endothelial, neuronal, and inducible) [3]. Underexpression or inhibition of DDAH decreases NOS activity and NO levels will decrease. Because of nitric oxide’s (NO) role in signaling and defense, NO levels in an organism must be regulated to reduce damage to cells [4]. This suggests that the rate-limiting step of this reaction is not the lid movement but is the actual chemistry happening to the substrate in the active site of DDAH [5].
The specific residues in the lid region are different in different organisms [3]. The only consistent similarity is a conserved leucine residue in this lid that function to hydrogen bond with the ligand bound to the active site [5]. Different isoforms from the same species can have differences in lid regions as well [3]. DDAH-2 has a negatively charged lid while DDAH-1 has a positively charged lid [3].
Active Site
The normal DDAH regulation mechanism depends on the presence of Cys249 in the active site that acts as a nucleophile in the mechanism [6]
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References
Tran CTL, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atherosclerosis Supplements. 2003 Dec;4(4):33-40. PMID:14664901 doi:10.1016/S1567-5688(03)00032-1
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:16698551 doi:10.1016/j.str.2006.03.006
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: 23097221 doi:10.1002/path.4127/full
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:17933965 doi:10.1152/ajpheart.00998.2007
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:PMC3945819 doi:10.1021/bi4015924
Stone EM, Costello AL, Tierney DL, Fast W. Substrate-assisted cysteine deprotonation in the mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa. Biochemistry. 2006 May 2;45(17):5618-5630. PMID:16634643 doi:10.1021/bi052595m
