Closing in on Roots of Depression
The thesis presents an expression system for the enzyme dopamine ß-monooxygenase (DBM) which is a potential target for therapeutic treatment.
About 15 per cent of the Danish population will experience depression at least once in life. It is known that depression, and also several other diseases including hypertension, Parkinson’s disease, and attention deficit hyperactivity disorder (ADHD), are linked to deficiency in the function of a key neurotransmitter, dopamine. Thus it has been suggested that enzymes related to dopamine conversion could be therapeutic targets. The thesis presents an expression system for one of these enzymes, dopamine β-monooxygenase (DBM). The lack of an efficient expression system for DBM has previously been a bottleneck for research on DBM as a potential target for therapeutic treatment.
DBM catalyzes the conversion of dopamine into another neurotransmitter, norepinephrine (NE). In the reaction, an oxygen atom from molecular oxygen is inserted into dopamine whereby NE and water are formed. As it contains copper (Cu) in the active site DBM is a metalloprotein. Metalloproteins are involved in a number of key biological processes. Besides copper, DBM requires ascorbic acid in order to be active.
NE is a neurotransmitter in the sympathetic nervous system where it regulates the cardiac contractility and a hormone in the neurosecretory cells. Since DBM regulates the balance between dopamine and NE, the activity level of DBM is decisive and makes DBM an important therapeutic target. Several DBM inhibitors have been tested, but have side effects and are non-responsive to certain populations. The efforts have been troubled with the lack of an efficient expression system for human DBM (hDBM).
In the project, hDBM was successfully expressed in mammalian cells in a stable glycosylated form. Further, hDBM was expressed in large enough amounts for structural characterization using intact protein mass spectrometric analysis and X-ray crystallography. The protein exists in two homo-oligomer forms; a dimer and a tetramer. Both forms were glycosylated, pure, stable, and displayed activity.
hDBM in both the dimer and the tetramer was set up for crystallization and the dimer formed crystals that diffracted to 2.9 Å. This is the first structure of DBM and the structure revealed new unexpected features and a completely new reaction mechanism for this class of enzymes.