Structural Insight of Dopamine β-Hydroxylase, a Drug Target for Complex Traits, and Functional Significance of Exonic Single Nucleotide Polymorphisms

Abstract

BackgroundHuman dopamine β-hydroxylase (DBH) is an important therapeutic target for complex traits. Several single nucleotide polymorphisms (SNPs) have also been identified in DBH with potential adverse physiological effect. However, difficulty in obtaining diffractable crystals and lack of a suitable template for modeling the protein has ensured that neither crystallographic three-dimensional structure nor computational model for the enzyme is available to aid rational drug design, prediction of functional significance of SNPs or analytical protein engineering.Principal FindingsAdequate biochemical information regarding human DBH, structural coordinates for peptidylglycine alpha-hydroxylating monooxygenase and computational data from a partial model of rat DBH were used along with logical manual intervention in a novel way to build an in silico model of human DBH. The model provides structural insight into the active site, metal coordination, subunit interface, substrate recognition and inhibitor binding. It reveals that DOMON domain potentially promotes tetramerization, while substrate dopamine and a potential therapeutic inhibitor nepicastat are stabilized in the active site through multiple hydrogen bonding. Functional significance of several exonic SNPs could be described from a structural analysis of the model. The model confirms that SNP resulting in Ala318Ser or Leu317Pro mutation may not influence enzyme activity, while Gly482Arg might actually do so being in the proximity of the active site. Arg549Cys may cause abnormal oligomerization through non-native disulfide bond formation. Other SNPs like Glu181, Glu250, Lys239 and Asp290 could potentially inhibit tetramerization thus affecting function.ConclusionsThe first three-dimensional model of full-length human DBH protein was obtained in a novel manner with a set of experimental data as guideline for consistency of in silico prediction. Preliminary physicochemical tests validated the model. The model confirms, rationalizes and provides structural basis for several biochemical data and claims testable hypotheses regarding function. It provides a reasonable template for drug design as well.

Characterization of tyramine β-hydroxylase, an enzyme upregulated by stress in Periplaneta americana

“…These processes may be involved in the tuning of the enzymatic activity of TBH and consequently may regulate OA level in insect body. The amino acid sequences of PaTBH isoforms are structurally organized according to the domain architecture of DBH, which contains three highly conserved domains, a DOMON domain, a copper type II ascorbatedependent monooxygenase N-terminal domain, and a copper type II ascorbate-dependent monooxygenase C-terminal domain (Aravind 2001, Kapoor et al 2011. The DOMON domain was initially described in secreted and membrane proteins in all phyla and was proposed to mediate a range of extracellular adhesive interactions (Aravind 2001).…”

“…Computational analysis showed that this domain is very diverse and is involved in heme and sugar recognition (Iyer et al 2007). More recently, structural analysis of hDBH suggested that the DOMON domain promotes tetramerization of the enzyme, while the Cu-monox-C domain controls dimerization (Kapoor et al 2011 Peptidylglycine a-monooxygenases also contain Cu 2C -monox domain, but in these latter, the DOMON domains are missing (Aravind 2001). TBH activity was analyzed using a new ELISA assay in nerve tissue of normal cockroaches as well as mechanical stressed animals.…”

In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies

“…By superimposing the model with PHM, two Copper metals were inserted manually based on the position of template. It could be assumed that Cu M binds to the Cterminal domain whereas Cu H binds to the N-terminal domain as described for PHM as well in case of crystal structure of human DβH [43,44]. For better understanding the insights of TβH, PDB file of the proposed model has been submitted as supplementary data.…”

“…A three-dimensional model of full-length human DβH protein was reported to reveals the structural insights using the template, peptidylglycine alphahydroxylating monooxygenase (PAM) [46]. After that, the crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution was reported very recently by Vendelboe and his groups, where two active site was found, one open active site same as for peptidylglycine alpha-hydroxilating monooxygenase and another close active site apart 4-5 Å from each other [44]. We've found two possible binding sites by analyzing all the residues involved in the binding of all ligands.…”

Dopamine β-Hydroxylase Inhibitors Enhance the Discriminative Stimulus Effects of Cocaine in Rats

“…Second, we assessed whether pretreatment with disulfiram would alter the discriminative stimulus effects of cocaine. To confirm that any observed effects with disulfiram were mediated exclusively by actions on DBH, we examined the effects of nepicastat, a highly selective DBH inhibitor that acts via competitive antagonism at the active site on the DBH protein and also increases basal and cocaine-induced increases in DA overflow (Stanley et al, 1997;Kapoor et al, 2011;Devoto et al, 2013). Finally, to determine whether the modulatory effects of disulfiram and nepicastat were mediated by excess DA originating from noradrenergic neurons, we assessed their impact on the cocaine-like stimulus effects of the selective NE transporter inhibitor reboxetine.…”