1H, 15N, 13C backbone resonance assignments of human soluble catechol O-methyltransferase in complex with S-adenosyl-l-methionine and 3,5-dinitrocatechol

Sylwia Czarnota,Nigel S Scrutton,Sam Hay,Nicola J Baxter,Matthew J Cliff,Jonathan P Waltho

doi:10.1007/s12104-016-9720-9

Sylwia Czarnota, Nigel S Scrutton + Show 4 more

Open Access

https://doi.org/10.1007/s12104-016-9720-9

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Abstract

Catechol O-methyltransferase (COMT) is an enzyme that plays a major role in catechol neurotransmitter deactivation. Inhibition of COMT can increase neurotransmitter levels, which provides a means of treatment for Parkinson’s disease, schizophrenia and depression. COMT exists as two isozymes: a soluble cytoplasmic form (S-COMT), expressed in the liver and kidneys and a membrane-bound form (MB-COMT), found mostly in the brain. Here we report the backbone 1H, 15N and 13C chemical shift assignments of S-COMT in complex with S-adenosyl-l-methionine, 3,5-dinitrocatechol and Mg2+. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97 % of all backbone resonances were assigned in the complex, with 205 out of a possible 215 residues assigned in the 1H-15N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS+ webserver is in good agreement with published X-ray crystal structures.

Highlights

Catechol O-methyltransferase (COMT, EC 2.1.1.6) is a ubiquitous bisubstrate magnesium-dependent enzyme found in plants, animals and microorganisms
It catalyses the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to one of the hydroxyl oxygen atoms in a catechol substrate (Mannisto and Kaakkola 1999)
There are two isoforms of human COMT: soluble cytoplasmic COMT (S-COMT), which is mainly intracellular, and a membrane-bound form (MB-COMT), which has a single-span helix contained within a 50 amino acid extension at the N-terminus

Summary

Biological context

Catechol O-methyltransferase (COMT, EC 2.1.1.6) is a ubiquitous bisubstrate magnesium-dependent enzyme found in plants, animals and microorganisms. It catalyses the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to one of the hydroxyl oxygen atoms (preferentially the 3-hydroxyl) in a catechol substrate (Mannisto and Kaakkola 1999). Genetic studies have demonstrated that both soluble and membrane-bound isoforms of human COMT are coded by a single gene, using two separate promoters, assigned to chromosome 22q11.2 (Tenhunen et al 1994). Several X-ray crystal structures have been solved for COMT enzymes from a range of organisms together with different substrate/ inhibitor complexes. S-COMT has a single domain α/βfolded structure with eight α-helices and seven β-strands. The active site is located on the outer surface of the enzyme and includes a SAM binding pocket and a substrate binding site situated in the vicinity of a bound catalytic Mg2+ ion (Vidgren et al 1994)

Protein expression and purification

NMR experiments

Findings

Resonance assignments and data deposition