A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization.
Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.
Nielsen
Michael S
MS
Department of Chemistry, Building 207, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
Petersen
Charlotte R
CR
Munch
Astrid
A
Vendelboe
Trine V
TV
Boesen
Jane
J
Harris
Pernille
P
Christensen
Hans E M
HE
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D020134:Catalytic Domain; D002645:Chickens / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014365:Tryptophan Hydroxylase / Q000737:chemistry* / Q000302:isolation & purification*
D014365:Tryptophan Hydroxylase