Functional domains of human tryptophan hydroxylase 2 (hTPH2).
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.
Carkaci-Salli
Nurgul
N
Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033-2360, USA.
Flanagan
John M
JM
Martz
Matthew K
MK
Salli
Ugur
U
Walther
Diego J
DJ
Bader
Michael
M
Vrana
Kent E
KE
D004790:Enzyme Induction; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012995:Solubility; D014365:Tryptophan Hydroxylase / Q000737:chemistry* / Q000378:metabolism
C494784:TPH2 protein, human; D014365:Tryptophan Hydroxylase