CATALYTIC DOMAIN OF CHICKEN TRYPTOPHAN HYDROXYLASE ISOFORM 1

Brookhaven Science Assoc-Brookhaven Lab, Upton NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Depression, eating disorders, schizophrenia, OCD (obsessive compulsive disorder) and many other neuropsychiatric disorders are presumably caused by imbalance in the level of the neurotransmitter serotonin in the brain.The iron-containing enzyme tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin and knowledge on the structure and function of this enzyme is thus important for the molecular understanding of the disorders mentioned above. The complete mechanism of TPH is not known.TPH is known to be notoriously unstable and difficult to purify, and only 5 crystal structures of the double truncated enzyme have been published. Our group has developed methods to purify stable solutions of several TPH variants, which have now been crystallized. One structure has already been solved and was published in Biochemistry in 2008 (PDB ID 3E2T). This was the first structure of any aromatic amino acid hydroxylase with the natural substrate bound. Now we have crystals of this same variant of TPH, with only substrate (the previous structure also contained an unnatural ligand from the crystallization solution) and with substrate and cofactor bound. Determination of these structures will allow us to investigate the structural changes which occur upon substrate and cofactor binding. Together with CD/MCD data collected at The Solomon Laboratory at Stanford University, the structures will give a complete picture of the structure/function relationship in TPH1. The crystals of TPH1 are between 40 and 100 um, clearly three dimensional and nicely shaped with smooth edges. The last crystal diffracted to a resolution of 1.90 [unreadable], however these new crystals appeared after shorter time (3 days vs. 6 months) so the actual resolution is not known.As we have several crystals for each of the two possible structures, a scouting process for the-best-in-the-lot using automounters would be preferable. The structures will be solved with molecular replacement using the previous structure (3E2T).