Proc. Nati. Acad. Sci. USAVol. 84, pp. 5530-5534, August 1987Biochemistry

Full-length cDNA for rabbit tryptophan hydroxylase: Functionaldomains and evolution of aromatic amino acid hydroxylases

(phenylalanine hydroxylase/tyrosine hydroxylase)

HERNAN E. GRENETT*, FRED D. LEDLEY, LORI L. REED, AND SAVIo L. C. WootHoward Hughes Medical Institute, Department of Cell Biology, Institute of Molecular Genetics, Baylor College of Medicine, Houston, TX 77030

Communicated by Hans Neurath, March 31, 1987

ABSTRACT A full-length cDNA for tryptophan hydroxy-lase was cloned from rabbit pineal body by screening anexpression library with antibody against rat phenylalaninehydroxylase, which crossreacts with rabbit tryptophan hydrox-ylase. Clones producing immunoreactive material containsequences homologous to, yet distinct from, phenylalaninehydroxylase. The rabbit cDNA hybridizes to mRNA in pinealbody and brainstem but not in liver. Comparison of the rabbittryptophan hydroxylase sequence with the sequences of phen-ylalanine hydroxylase and tyrosine hydroxylase demonstratesthat these three biopterin-dependent aromatic amino acidhydroxylases are highly homologous, reflecting a commonevolutionary origin from a single primordial genetic locus. Thepattern of sequence homology supports the hypothesis that thecarboxyl-terminal two-thirds of the molecules constitute theenzymatic activity cores, and the amino-terminal thirds of themolecules constitute domains for substrate specificity.

Tryptophan hydroxylase [TrpOHase; L-tryptophantetrahy-dropteridine:oxygen oxidoreductase (5-hydroxylating), EC1.14.16.4] is a rate-limiting enzyme in serotonin biosynthesisand has an important physiological role in the central nervoussystem, the gastrointestinal tract, and vascular homeostasis(1-6).TrpOHase is one member of a family of biopterin-depen-

dent aromatic amino acid hydroxylases that includes phen-ylalanine hydroxylase (PheOHase, EC 1.14.16.1) and tyro-sine hydroxylase (TyrOHase, EC 1.14.16.2) (7-10). Theseenzymes catalyze a mixed oxidation reaction that consumesa reduced biopterin cofactor and molecular oxygen for thehydroxylation of an aromatic amino acid substrate. Theseenzymes share many physical and biochemical properties (9,11-13), and antibodies to PheOHase crossreact with Tyr-OHase and TrpOHase (7, 8, 14).

Purified TrpOHase has been reported to have a molecularweight between 52,000 and 60,000 (15, 16). TrpOHase is amultimer of identical subunits that can be activated by phos-phorylation, phospholipids, and partial proteolysis (7, 8, 15-18).

Cloning and sequencing of cDNAs for PheOHase (19-22)and TyrOHase (23) have been reported. Here we describe thecloning and sequencing ofcDNA for rabbit pineal TrpOHaseand analysis of the structural, functional, and evolutionaryrelationships of these three enzymes.

MATERIALS AND METHODSIdentification of TrpOHase Immunoreactive Protein and

Assay of TrpOHase Activity. Cytoplasmic extracts fromrabbit pineal body were fractionated by NaDodSO4/PAGE.Proteins were electrophoretically transferred to nitrocellu-lose paper and probed sequentially with goat anti-rat Phe-

1 2 3 4

__u

Ezi.

a)0

0I-

0-

10IgG, Ag

FIG. 1. Identification of TrpOHase in cytoplasmic extracts ofrabbit pineal body by anti-PheOHase antibody. (Left) Immunoblotshowing crossreactive material identified by affinity-purified goatanti-rat PheOHase antibody. Lane 1: rat liver, showing PheOHaseimmunoreactivity (arrow). Lane 2: rabbit liver. Lane 3: mouse raphenucleus, showing immunoreactivity with TyrOHase. Lane 4: rabbitpineal body, showing immunoreactivity with TrpOHase. (Right)Inhibition of TrpOHase activity in rabbit pineal extracts with goatanti-rat PheOHase antibody. Activity was assayed as conversion of['4C]tryptophan to 5-hydroxy['4C]tryptophan. Inhibition was as-sayed by preincubating the reaction mixture with goat anti-ratPheOHase antibody (o) or preimmune goat IgG (e).

OHase antibody (affinity-purified on a rat PheOHase column,ref. 19) and 1251-labeled porcine anti-goat IgG. The TrpOHaseassay measures conversion of [14C]tryptophan to 5-hydroxy-L-[14C]tryptophan. Assay mixtures (0.1 ml) contained['4C]tryptophan (4 x 105 cpm; New England Nuclear, >500mCi/mmol; 1 Ci = 37 GBq), 0.2 M Tris acetate (pH 7.65), 0.15M KCI, 6 units of bovine liver catalase (Sigma), cytoplasmicextract (500 Ag of protein) in 0.15 M KCI/2 mM dithiothre-itol/10, M 6-methyltetrahydropterin and were incubated for1 hr at 37TC. Reaction products were separated by chroma-tography on Whatman 3MM paper in butanol/acetic acid/water (60:15:25, vol/vol). For quantitation, radioactivitycorresponding to 5-hydroxy-L-tryptophan was measured byscintillation counting. Net counts were calculated as themean of duplicate samples minus reaction blanks run withoutcofactor. Inhibition by anti-PheOHase antibody was assayedby preincubation of antibody with enzyme sample for 15 minat 250C followed by centrifugation for 15 min in an Eppendorfmicrocentrifuge. PheOHase activity was assayed as de-scribed (22).

Construction of cDNA Library and Screening for Trp-OHase. A cDNA library was constructed by the method ofYoung and Davis (24, 25) from rabbit poly(A)+ RNA from 500pineal bodies (Cappel). The library contained 800,000 inde-

Abbreviations: TrpOHase, tryptophan hydroxylase; PheOHase,phenylalanine hydroxylase; TyrOHase, tyrosine hydroxylase.*Present address: Department of Cell Biology and Anatomy, Uni-versity of Alabama-Birmingham, 1918 University Blvd., Birming-ham, AL 35294.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Biochemistry: Grenett et al. Proc. Natl. Acad. Sci. USA 84 (1987) 5531

1S 30 45 60 75CC GAG TCC CGC CGC ACC AGG CTC CCC TCA AGO ATT TGA TCG CCC TCG COG CGG CCG CCC CAG CCC ACC CTC CTG GCA CCC TGT CAT CTA

90 105 120 135 1S0 165TAG TTT CAT ACT TTA GTT TTA GAG AAT TAC TCA AAA CTC ATC ATC ATT CAA GAT AAT AAA GAG AAC AAA GAC CAT TCT TTS GAA AGA GGA

Not Ile Glu Asp Asn Lys Glu Asn Lys Asp His Ser Liu Clu Arg Gly

10 195 210 225 240 255AGA GCA ACT CTC ATT TTT TCC TTA AAC AAT GMA GTT GCA GGA CTT ATA AAA GCA CTG AAA ATC TTC CAG GAG AA CAT CTC AAC CTG TTAArg Al& Thr Lou IIe Phe Ser Lou Lys Asn Glu Val Gly Gly Lou Ii1 Lys Al& Lou Lys Ile Ph. Gln Glu Lys His Val Asn Lou Lu

270 215 300 315 330 345CAT ATT GAG TCC CCA AAA TCT AAG AGA ACA AAC TCA MA TTT GAG ATT TTT OTT GAC TOT GAT ACC AAC ACA CAA CAA TTG AAT OAT ATTHis 1i Giu Ser Arg Lys Ser Lys Arg Arg Asn Ser Glu Phe Glu Ile Ph. Val Asp Cys Asp Thr Asn Arg Clu Gln Lou Asn Asp II1

360 375 390 405 420 435TTT CAT CTT CTS AAG TCT CAT ACC AAT GTT CT? TCT CTC ACT CCA CCA GAT AAT TTT ACT ATG AAG GAA GAA OGA CTG GAG AGT OTT CCTPh. His Lou Lou Lys Ser His Thr Asn Val Lou Ser Va1 Thr Pro Pro Asp Asn Phe Thr not Lys Glu Olu Gly Lou Clu Ser Val Pro

450 465 480 495 510 525TGO TTT CCA AAC AAG ATT TCA GAC CTG GAC CAT TGT OCT AAC CCA CTT CTG ATG TAT GCA TCT GAG CTA OAT GCA GAC CAC CCT CCC TTCTrp Phe Pro Lys Lys Ile Ser Asp Lou Asp His Cys Ala Asn Ar; Val Lou Hot Tyr Gly Ser Glu Lou Asp Al& Asp His Pro Gly Phe

540 555 570 585 600 615AAA GAT AAT GTC TAC CCT AAA AGA CGA AAG TAC TTT GCA GAC TTC GCT ATG AOC TAT AAA TAT OGA GAC CCC ATT CCT AAG GTT GAA TTCLys Asp Ann Val Tyr Arx Lys Arg Arg Lys Tyr Phe Ala Asp Lou Al& Nat Ser Tyr Lys Tyr Cly Asp Pro Ile Pro Lys Val Glu Phe

630 645 660 675 690 705ACG CAA GAG GAO ATT AAG ACC TOO CGA ACC CTA TTC COG GAO CTC AAC AAA CTC TAT CCG ACC CAT OCT TGC AGA GAG TAT CTC AAA AATThr Clu Glu Glu Ile Lys Thr Trp Gly Thr Val Phe Arg Glu Lou Ann Lys Loeu Tyr Pro Thr His Al& Cys Arg Glu Tyr Lou Lys Asn

720 735 750 765 780 795TTA CCT CTC CTT TCC AAT GAT TOT GGA TAT COO GAA GAC AAT ATC CCA CAC CTO CAA GAT ATT TCA AAC TTT TTA AAA GAG CGC ACA GGTLou Pro Lou Lou Ser Asn Asp Cys Gly Tyr Arg Glu Asp Asn Ile Pro Gln Lou Olu Asp I1. Ser Asn Ph. Lou Lys Glu Arg Thr Gly

*10 825 840 855 870 385TTT TCC ATT CGT CCT GTC GCT GOT TAC TTA TCA CCA AGA GAT TTC TTA TCA GOT TTA CCC TTT CGA OTT TTT CAC TGC ACT CAA TAT GTGPh. Ser Ile Arg Pro Val Al& Cly Tyr Lou Ser Pro Arg Asp Phe Lou Ser Gly Lou Al& the Arg Val Phe His Cys Thr Gin Tyr Val

900 915 930 945 960 975AGA CAC ACT TCA GAC CCC TTC TAT ACC CCA GAO CCO GAT ACC TOC CAT GAA CTC TTA GOT CAC GTT CCC CTT TTG GCT GAG CCA AGT TTTAr; His Ser Ser Asp Pro Phe Tyr Thr Pro Glu Pro Asp Thr Cys His Glu Lou Lou Gly His Val Pro Lou Lou Ala Glu Pro Ser Phe

990 1005 1020 1035 1050 1065OCT CAG TTC TCC CAA GAA ATT GCC CSC CCT TCC CTT CGA OCT TCA GAG GAG CCT OTT CAA AAA CTG CCA ACG TGC TAC TTT TTC ACT GTCAla Gln Ph. Sor Gln Glu Ile Gly Lou Al& Ser Lou Gly Al& Ser Glu Glu Ala Val Gln Lys Lou Al& Thr Cys Tyr Phe Phe Thr Val

1060 1095 1110 1125 1140 1155GAO TTT GCT CTA TOT AAA CAA GAT CGA CAG TTA CGA GTC TTC CCC CCT CCC TTA CTT TCT TCT ATC ACT GAA CTC AAA CAT OTO CTT TCTClu Pho Cly Lou Cys Lys Gin Asp Gly Gin Lou Arg Val Ph. Cly Al& Gly Lou Lou Ser Ser Ile Ser Glu Liu Lys His Val Lou Ser

1170 1lls 1200 1215 1230 1245GGA CAT CCC AAA GTA AAC CCT TTT GAT CCC AAC ATT ACC TAC AAA CAA GAA TCC CTC ATC ACA ACT TTT CAC OAT CTC TAC TTT OTA TCTCly His Ala Lys Val Lys Pro Phe Asp Pro Lys Ile Thr Tyr Lys Gln Glu Cys Lou Ile Thr Thr Phe Gln Asp Val Tyr Ph. Val Ser

1260 1275 1290 1305 1320 1335GAA AGC TTT CAA GAT CCA AAC GAG AAG ATC A"A GAA TTT AAA AAA ACA ATT AAG COT CCC TTT CCA CTC AAA TAT AAT CCC TAC ACA CGAClu Sor Phe Clu Asp Al& Lys Clu Lys not Arg Glu Phe Lys Lys Thr Ii. Lys Arg Pro Ph. Cly Val Lys Tyr Asn Pro Tyr Thr Arg

1350 1365 1380 1395 1410 1425AGC ATT CAG ATC CTG AAA GAC CCC AAA AGC ATA ACG AAT CCC ATG AAC GAG CTG CGO CAT GAT CTT GAC OTT GTC AGC GAC GCC CTT GGGSer Ile Gln Ile Lou Lys Asp Ala Lys Ser Ile Thr Asn Ala Not Asn Olu Lou Arg His Asp Lou Asp Val Val Sor Asp Ala Lou Gly

1440 1455 1470 1485 1500 1515OG CTC AGC AGO CAC COT AGT OTC TGA CCC CTG CCA CTC CTA ACT CAG ACA CCA TCT GAG CAT CGA TTC AOA GOC CTT CGT 0GG CCA GCC

Lys Val Ser Arg Gln Lou Ser Va1 **-

1530 1545 1560 1575 1590 1605CTO CTT TTC TTG GAO ACC TGA TCA TCC AAG OAT AOC CCC TTC TOG CTA AAT OAT ACT CTA ATC CCA TTT CTT AAT GAA TCA CTA GGC TAA

1620 1635 1650 1665 1680 1695AAC TOC ACC TGC ATA CTT TAT CTC CTA ATC ATT TTG TTT TTG CTA ATS OTT TAO TGA AAT ACA ATT CAC ATA CCA TAC AAT TTA CTC ACC

1710 1725 1740 1755 1770 1735ACC CAT TTT TAG TAG TAA ATT OTT TGA CCC AAT CGA TOG AAC TAO TCT COG CCT ATT TCC TCC AAT TCT TCC TGA ATA AGA TOO ATT TTA

1800 l8ls 1830 1845 1860 1875TGA TTA TTT AGO TCT CTA TGA GTA TTA TAG AGC TCT GAC TCA TOT TOT TAC CTG AGA TCC CTT TAT TTA TAA AGT AT? TOC TAC TTA CTA

1890 1905 1920 1935 1950 1965CCG AAT TTG TCC CAA GCT CAG TGA CAT TCA CAC ACC CAA CTT OAT AGA TCA OTT OTT TTA TAC TCC TAO CAT GAG CAA AAT TAT AOA CGT

1980 1995 2010 2025 2040 2055CTT TAT ACA CAO CCC ATA CCT ACT AAC ACT TOO TAO TCT ACT TTC AAG TOT ACT ATT TTA CAG CAA MT AAO ACT AGC TAT CTC GAA AGO

2070 2085 2100 2115 2130 2145TTG ATT COO CAT AAG CCT TCA CCC TAA TAA TTT TAG GCG CCC CTT TCC AAA TAA CTA TTC TTG AAA CTC ACT TTC CTT TCT ATG GAC ATC

2160 2175 2190 2205 2220 2235ATA TAA TSC TOG CTT TCT CCT AGA TTA TCT CTC TTC CTC CTC AGT ACA AGA CGA AAM TOG TAT CGT 0CC ACC TOO AM 0CC TTG TAC TCA

2250 2265 2280 2295 2310 2325CAC ATS CAT CCC CGA GAT CTT CTA ACT COO ATT ATC CTC TGG CTA OCA GGA CGT AMT CTG CTC AMA CGC AGO GTT CAA ACT GAG ATA AGA

2340 2355 2370 2385 2400 2415GAA CCA CAA ACT CMA ACT TCO AAM AGC ATG CCA AGC ACT TTA TCT CCA COO TAA GCA OTG AGO GCA GAA OAT CCC GAC AMC COG AGO AAM

2430TOG ATC TOG AM

FIG. 2. Complete nucleic acid sequence and predicted amino acid sequence of rabbit TrpOHase (clone prbTRH479).

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53Proc. Na/I. Acad. ASci. USA 84 (1987)

pendent plaques (92% recombinants). Clones producing im-munoreactive protein were identified by probing sequentiallywith a 1:2000 dilution of goat anti-rat PheOHase and 1251_labeled porcine anti-goat IgG in BLOTTO (25, 26). ThecDNA library was rescreened with the cDNA insert of cloneprbTRH401 according to published methods (27). Mini phagelysates were prepared as described (28).DNA Sequencing. DNA sequences were determined by the

dideoxy method (29). EcoRI fragments were subcloned intoM13mpl8 phage and sequenced in both orientations. The1.7-kilobase EcoRI fragment of prbTRH479 was sequencedby sequential deletion, using the method of Henikoff (30).Sequence analysis was performed using the on-line facilitiesof the Protein Identification Resource (PIR) at the NationalBiomedical Research Foundation (31, 32).RNA Blot Hybridization Analysis. Total RNA was electro-

phoresed in formaldehyde/agarose gels (33), transferred tonitrocellulose (34), prehybridized, and hybridized with 32p_labeled insert from prbTRH479 as described (19). The finalwash was 15 mM NaCl/1.5 mM sodium citrate, pH 7.0/0.5%NaDodSO4 at 680C.

RESULTS

Identification of Rabbit TrpOHase cDNA Clones. We in-vestigated whether monospecific antibody against ratPheOHase would react with TrpOHase of rabbit pineal body.Immunoblotting identified a single band in rabbit pinealextract at an apparent molecular weight consistent with thesize expected for TrpOHase (Fig. 1 Left). No PheOHaseactivity was detectable in rabbit pineal extract (data notshown). TrpOHase activity was present and was inhibited>90% by addition of anti-rat PheOHase antibody (Fig. 1Right), indicating that the immunoreactive material identifiedby immunoblotting was TrpOHase. Normal goat IgG did notinhibit TrpOHase.A rabbit pineal cDNA library was screened with anti-rat

PheOHase antibody. One positive clone was detected from800,000 plaques and was used as a probe to rescreen therabbit pineal cDNA library. Four clones were plaque-purifiedand characterized by restriction mapping.Sequence of Rabbit TrpOHase mRNA and Protein. The

longest cDNA clone, prbTRH479, contained 2441 bases. Thisclone contains a single long open reading frame starting withan ATG at position 132 and ending with a TGA stop codon at1464 (Fig. 2). The continuous open reading frame comprises1332 bases coding for a protein with 444 amino acids and apredicted molecular weight of 51,007. The clone contains a131-base 5' untranslated region and a 75-base 3' untranslatedregion. The nucleotide and predicted amino acid sequence ofrabbit TrpOHase are homologous to the sequence of a shortcDNA clone for rat TrpOHase, isolated from a rat pineallibrary by screening with a monospecific antibody against ratTrpOHase, reported by Darmon et al. (35). Of 94 amino acidresidues reported for rat TrpOHase, only five are different inthe predicted sequence of rabbit TrpOHase and three of thedifferences are highly conservative (Lys-*Arg or Ile->Val).TrpOHase mRNA in Rabbit Pineal Body and Brainstem.

Blot hybridization analysis of mRNA from different rabbittissues was performed using prbTRH479 as a probe andstringent hybridization and wash conditions (Fig. 3). Hybrid-ization was observed to mRNA from rabbit pineal body andbrainstem (lanes 1 and 4) but not to mRNA from muscle, lung,kidney, or liver (lanes 2, 3, 5, and 6).Homology to PheOHase and TyrOHase. The predicted

amino acid sequence of rabbit TrpOHase is highly homolo-gous to the sequences of PheOHase and TyrOHase (Fig. 4).TrpOHase and PheOHase are 53% homologous (230/434residues) with insertion of a single gap of four amino acidsnear the amino terminus. TrpOHase and TyrOHase are

1 2 3 4 b 6

28S

TrpOHasc

18S

FIG. 3. Blot analysis of expression of TrpOHase mRNA. Lanes:1, pineal body; 2, skeletal muscle; 3, lung; 4, brainstem; 5, kidney;6, liver. Positions of 28S and 18S rRNA are indicated.

slightly less homologous (48%; 209/432 residues) with theinsertion of two gaps. Many of the nonhomologous residuesrepresent highly conservative changes such as Ile-*Leu,Ser--Thr, Tyr-*Phe, Gln- Glu, and Asn--Asp.There are several significant differences in the primary

structures of these three molecules. The most pronounceddifference is that a long stretch between amino acids 65 and105 of TrpOHase exhibits little or no homology with eitherPheOHase or TyrOHase. Also striking is the lack of homol-ogy between four of the nine cysteine residues in TrpOHaseand cysteine residues in PheOHase or TyrOHase.

DISCUSSION

This report describes the cloning and sequence of a full-length cDNA for TrpOHase from rabbit pineal body. Severalobservations demonstrate that these clones code for authen-tic TrpOHase. First, the sequence of the clone reported herematches the sequence of an incomplete rat TrpOHase cloneobtained from a rat pineal library by screening with antibodyraised against rat TrpOHase (35). Second, RNA blot analysisusing high-stringency hybridization and wash conditionsshowed that the rabbit TrpOHase clone hybridizes to mRNAfrom rabbit pineal body and brainstem but not liver, which isconsistent with the tissue-specific distribution of TrpOHaseand unequivocally distinguishes this clone from PheOHase(3, 4).The molecular weight of TrpOHase has been variously

reported to be between 52,000 and 60,000 (15, 16). Themolecular weight predicted for rabbit TrpOHase by sequenceanalysis is 51,007. The first ATG in the cDNA is at position132 and is preceded by two in-frame TGA stop codons atpositions 27 and 36. TrpOHase is thus shorter thanPheOHase at the amino terminus by 17 residues, and thoughit is 5 residues longer at the carboxyl end, its predictedmolecular weight is lower than that of PheOHase.TrpOHase is highly homologous to both PheOHase and

TyrOHase, though there is considerably more homologybetween TrpOHase and PheOHase than between TrpOHaseand TyrOHase (Table 1). All three molecules exhibit sub-stantial homology surrounding five common cysteine resi-dues at the core of these sequences (Fig. 4) and less homologyat the amino-terminal end. TrpOHase is 60% identical toPheOHase and TyrOHase from amino acid 100 to thecarboxyl terminus (Table 1). The amino-terminal sequence ofTrpOHase, residues 1-104, is

Biochemistry: Grenett et al. Proc. Natl. Acad. Sci. USA 84 (1987) 5533

PAM --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---- --- --- --- --- ---TRH --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -- - --- --- --- --- ---TYH 1 Met Pro Thr Pro Ser Ala Pro Ser Pro Gln Pro Lys Gly Phe Arg Ar; Ala Val Ser Glu Gln Asp Ala Lys Gln Ala Glu Ala Val Thr

PAH 1 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu GlyTRH - -- - -- --- --- - -- --- --- --- -- - --- --- -- - --- --- - -- --- --- --- --- - -- - -- - -- - -- --- - -- - -- - -- - -- - -- -- -TYH 31 Ser Pro Arg Phe Ile Gly Arg Arg Gln Ser Leu Ile Glu Asp Ala Arg Lys Glu Arg Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala Val

PAM 13 Arg Lys Leu Ser Asp Phe Gly Gin Glu Thr Ser Tyr Ile Glu Asn Cys Asn Gin AsnFG Ala Ile Ser Leu Ile Phe Ser Leu LysTRH 1 --- --- --- --- --- Met Ile Glu Asp Asn Lys Glu Asn Lys Asp His Ser Leu Glu ArgG[y Arg Ala Thr Leu Ile Phe Ser Leu Ly]TYH 61 Ala Ser Ser Glu Pro Gly Asn Pro Leu Glu Ala Val Val Phe Glu Glu Arg Asp Gly Asn Aa Val Leu Asn

PAM 43 --- GluG u a1 GI Ala Leu Ala ValLou Arg Leu Gu u Asn ASP VAs n Leu Thr His Ile Glu Ser Arg Pro ArgTRH 26 --- Asn Glu Val Glyj Gly Leu Ile Lys A a Leu Lys Ile Phe Gin i Lys His Val Asn Leu Leu His Ile Glu Ser Arg Lys LysTYH 91 Gly Thr Psro Ser Ser Leu Ser Arg Ala Lys Val Phe Glu r Phe Glu A a Lys Ile His His Lou GIu T r Ar Pro a Gln

PAH 72 Leu Lys Lys Asp TyrG Ph.e P --- --- --- --- His Leu Asp Lys Arg Ser Leu Pro Ala Leu Thr Asn Ile Ile Lys IleTRH 55 rg Arg Asn Ser Phe Ile Ph Val Asp Cys Asp jAsn ArgTGl Gln Leu Asn Asp Ile Phe His Leu Leu Lys Ser His ThrTYH 121 J --- --- Leu Ala Gly Ser Pro His LeuLG Tyr Phe Val Arg Phe Glu Val Pro Ser Gly Asp Leu Ala

PAR 99 Leu Arg His Asp Ile Gly Ala Thr Val His Glu Leu Ser Arg Asp Lys Lys Lys Asp Thr Val Pro Trp Phe Pro Ar r Gln GluTRH 85 Asn VaI Leu Ser Val Thr Pro Pro Asp Asn Phe Thr Met Lys Glu Glu Gly Leu Glu Ser Val Pro Trp Phe Pro Lys Ile Ser AspTYH 144 Ala Lou Leu Ser Ser Val Arg Arg Val Ser Asp Asp Val Arg Ser Ala Arg Glu Asp Lys Val Pro Trp Phe Pro Arg Ser Glu

PAM 129 Leu Asp Ar Ph AlaAg GlnI e u Seryr GlyAla Giu Leu Asp Ala Asp His Pro Gly Phe Lys Asp ProVal TyrA AlaATRH 115 Leu Asp His Ala Asn Arg Lou Met Gly|Ser Glu Leu Asp Ala Asp His Pro Gly Phe Lys Asp Asn Val Tyr Arg Lys ArgTYH 174 Leu Asp Lys H s His Leu Val Thr Lys Phe Asp Pro sp Lou AsprLou Asp His Pro Gly Ph. GinVal Tyr Ar; GinAr

PAH 159 Arg Lys Gln Phe Ala Asp Ile Ala Tyr Asn yr Arg His Gly Gin Pro Ile Pro Arg;Val GITyrLrGluGluG lp7RH 145 Arg Lys Tyr Phe Ala As Leu Ala Met Ser Tyr Lys Tyr Gly Asp Pro Ile Pro Lys Val Glu Phe Glu Glu Glu e L s Thr TrpTYH 204 Arg Lys Leu Glu Ile AlaPhe Gln|Tyr Lys His Glu Pro Ile Pro HisTValGluyrGTu I Al

PAH 189 [Gly Thr Val Pe Lys Thr Lou Lys Ser Leu Tyr Lys Thr His Ala Cys Glu Ty Asn His Ile Phe Pro Leu Leu Glu Lys Tyr CysTRH 175 Gly Thr Val Phe Arg Glu Leu Asn Lys Leu Tyr Pro Thr His Ala Cys Arg Glu u Lys Asn Leu Pro Leu Leu Ser L s Tyr CysTYH 234 Lys lajyr Val Thr Lou Lys Gly Leu Tyr Ala Thr His Ala Cys Arg Glu Lu Glu Gly Phe Leu Leu Glu Arg;Tyr Cys

PAH 219 in Lou Giu Asp Val Gin Ph. Lou Gin Thr Cys Thr Gly Pho Arg Leu Arg Pro Val Ala GlyTRH 205 G1Y Tyr Arg Glu Asp Asn Ile Pro Gln Leu Glu Asp Ile Ser Asn Phe Leu Lys Glu Ar; Thr Gly Phe Ser Ile Arg Pro Val Ala GlyTYH 264 GY Tyr Arg Glu Asp -ril Pro Gn Lu Glu Asp Val Arg Phe Leu Lys Glu Arg Thr Gly Phe Gln Leu Arg Pro Val Ala Gly

PAH 249 Leu Leu Ser Ser Ar; Asp Phe Leu Gly Gly Leu Ala Phe Arg Val Phe His Cys Thr Gin Tyr Ile Arg His Gly r Lys Pro MetTRH 235 Tyrt Leu Ser Pro Arg Asp Phe Leu Ser Gl Leu Ala Phe Ar; Val Phe His Cys Thr Gln Tyr Val Arg His Ser Ser|Asp Pro Phe.TyrTYH 294 LeuoLeu Ser Ala Arg Asp Phe Leu Ala Ser Leu Ala Phe Arg Val Ph.eGin Cys Thr GIn Tyr Ile Arg His Ala SerProHMet his

PAH 279 Thr Pro Glu Pro Asp IleCys His Glu Leu Leu Gly His Val Pro Leu Phe Ser Asp Arg;Ser Ph. Ata Gln Phe Ser Gin Glu Ile Gly7RH 265 Thr Pro Glu Pro Asp Thr Cys His Glu Leu Leu Gly His Val Pro Leu Leu Al Glu Pro Ser Ph. A a Gln Phe Ser Gln Glu Ile GlyTYH 324 Pro Glu Pro Asp Cys ys His Glu Leu Leu Gly His Val Pro Met Lu Aa sp Arg Thr Phe Ala Gln Phe Ser Gin Asp-Il Gy

PAH 309 Leu Ala Ser Leu Gly Ala Pro AspG Tyr Ile Glu Lys Leu Ala Thr Ile[y Trp| Phe Thr Val Glu Phe Gly Leu Cys Lys Gln GlyTRH 295 Leu Ala Ser Leu Gly Ala Ser GlutGlu Ala Val Gln Lys Leu Ala Thr Cys Tyr Ph.e Phe Thr Val Glu Phe Gly Leu Cys Lys Gln AspTYH 354 Leu Ala Ser Leu Gly Ala Ser AspG Glu Ile Glu LosLuSorThr Val Tr Trpl Phe Thr Val Glu Phe Gly Leu Cys Lys Gln Asn

PAH 339 Asp Ser Ile Lys Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly Glu Leu Gln Tyr Cys Leu Ser Glu Lys Pro Lou LeuPro LeuTRH 325 JGT7j Gln LeujArg Val Phe.Gly Ala Gly Leu Leu Ser Ser Ile Ser Glu Leu Lys i |Val Leu Ser Gly His Ala LyssPVaTYH 384 GluyjGu Lys Ala TyrG Ala Lu S eriTyr Gly-Glu LeuL e uSer Leu Ser Glu Glu Pro 1 VaArgAlaAr

PAH 369 Glu Leu Glu Lys r Ala IleGin Asn Tyr Thr Val Ph. Gin Pro Lou T Tyr V A Glu Sor Pho Asn Asp Ala Lys GlTRH 355 ASP Pro Lys Ile Tyr Lys Gln| Glu Cys Leu Ile. Thr Tr Phe Gln Asp Va Tyr Phe Val Ser Glu Ser Pheo Glu Asp Ala Lys GluTYM 414 Asp Pro Asp Thr Ala Ala Val Gin Pro Tyr Gin Asp n Thr TyrGn Proal Tyr Phe Val Ser Glu Ser Phe Asr Asp Ala Lys Asp

PAH 399 Lys Val Ar; Asn Ala Ala Thr le Pro Arg Pro Phe Ser ; yr Asp Pro Tyr Thr Gln Arg; Glu Val Leu Asp Asn Thr7PH 385 Lys Met Arg Glu Ph. Lys LysThr '.le Lys Arg Pro PheGly Lys Tyr Asn Pro Tyr Thr Arg Ser Ile Gln Ile Leu Lys Asp Ala7yl 444 LJ Leu Arg Asn Tyr Ala Ser Ar; Il Gln Arg Pro Phe SerrVal LysPe Asp|Pro Tyr Thr Leu Ala Ijl Asp Val Leu Asp Ser Pro

?All 429 Gln Gln Leu Lys Ile Leu Ala Asp Ser Ile Asn Ser Glu Ile Gly Ile Leu Cys Ser Ala Leu G1in s Ile Lys *RPH 415 Lys SerrTiTe|Thr Asn Ala Met Asn Clu Leu Arg His Asp Leu Asp Val Val Ser Asp Ala Leu GlyLysI Val Ser Arg Gln Leu Ser Val-ri 474 His Thr 1 Gln Arg Ser Leu Glu Gly Val Gln Asp GluLe His Thr Leu Ala His Ala Leu Ser Ala Ile LJ

FIG. 4. Alignment of amino acid sequences for human PheOHase (PAH) (20), rabbit TrpOHase (TRH), and rat TyrOHase (TYH) (23). Boxesillustrate residues in PheOHase or TyrOHase that are identical to those of TrpOHase.

contributes to regulation and specificity (9, 10). The homol- calculated by comparing the rate of divergence of PheOHaseogy of TrpOHase, PheOHase, and TyrOHase suggests that from different species (human, rat, mouse) with the apparentTrpOHase will exhibit analogous domains. divergence between PheOHase, TyrOHase, and TrpOHaseThe extensive homology among these three proteins indi- (32). These calculations assume a constant rate of conserv-

cates that these enzymes evolved by duplication and diver- ative evolution in which sequence divergence occurs only bygence from a common ancestral gene (9, 10, 31). A rough the accumulation of point mutations measured in PAMsestimate of when this duplication may have occurred can be (accepted point mutations). The rate of divergence of the

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5534 Biochemistry: Grenett et al.

Table 1. Homology and evolutionary distance between aromaticamino acid hydroxylases

Human Rat Rabbit RatPheOHase PheOHase TrpOHase TyrOHase

Human PheOHase 8/6 49/41 53/39Rat PheOHase 9 49/41 55/40Rabbit TrpOHase 78 78 58/43Rat TyrOHase 89 93 103

Values above the diagonal are percent differences for optimallyaligned sequences (complete sequence/residues 105-144). Valuesbelow the diagonal are numbers of accepted point mutations (PAMs),a measure of evolutionary distance (31, 32) between the completesequences.

complete PheOHase sequences is =10 PAMs/100 millionyears (9) (Table 1). The evolutionary distance betweenPheOHase and TyrOHase is calculated at -750 million years,and the evolutionary distance between PheOHase andTrpOHase is calculated at =600 million years (Fig. 5).Alternative modes of evolution, such as exon rearrangementor gene conversion, may be suggested by the fact that themutation-acceptance rate is not uniform throughout thesemolecules (Table 1). Local variations in the mutation-accep-tance rate may also reflect the preservation of sequencesessential for enzyme activity.

It has been suggested that other enzymes involved incatecholamine biosynthesis, such as dopamine P-hydrox-ylase or phenylethanolamine N-methyltransferase, might bestructurally homologous to the pterin-dependent aromaticamino acid hydroxylases (38, 39). It has also been suggestedthat there may be several -forms of TrpOHase representingthe products of distinct genetic loci (40). These last specula-tions arise from the different kinetic properties of TrpOHasefrom different organs. The present work demonstrates thatthe TrpOHase cDNA from rabbit pineal body hybridizes tomRNA from pineal body and brainstem under extremelystringent conditions. We observed only a single mRNAspecies in these experiments and in the cDNA library, nottwo species as reported by Darmon et al. (35). We did notanalyze mRNA from chromaffin cells, ganglia, histiocytes, ormast cells, which are also reported to express Tr'pOHaseactivity, and cannot address the possibility that they mayhave a distinct hydroxylase.

We thank Dr. David George of the National Biomedical Research

PheOHaserat human

Trp0HaseTyrOras

FIG. 5. Schematic evolutionary tree of rqmmalian aromaticamino acid hydroxylase superfamily, based pn data in Table 1.Approximate 100-, 500-, and 750-million-year intervals are shown.

Foundation for construction of evolutionary trees, Ms. Whei MeiHuang for expert technical assistance, and Ms. Cyndi Hammonds forpreparation of the manuscript. This work was partially supported byNational Institutes of Health Grant HD17711. S.L.C.W. is anInvestigator and F.D.L. an Assistant Investigator of the HowardHughes Medical Institute.

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