Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in the Vmax value and the Km values for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.

Probing the relative timing of hydrogen abstraction steps in the flavocytochrome b2 reaction with primary and solvent deuterium isotope effects and mutant enzymes.

Flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate O-H and C-H bonds by the wild-type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The (D)V(max) and (D)(V/K(lactate)) values are both 3.0 with the wild-type enzyme at pH 7.5 and 25 degrees C, increasing to about 3.6 with the flavin domain and increasing further to about 4.5 with the D282N enzyme. Under these conditions, the (D20)V(max) values are 1.38, 1.18, and 0.98 for the wild-type enzyme, the flavin domain, and the D282N enzyme, respectively; the (D20)(V/K(lactate)) values are 0.9, 0.44, and 1.0, respectively. The (D)k(red) value is 5.4 for the wild-type enzyme and 3.5 for the flavin domain, whereas the solvent isotope effect on this kinetic parameter is 1.0 for both enzymes. The V(max) values for the wild-type enzyme and the flavin domain are 32 and 15% limited by viscosity, respectively. In contrast, the V/K(lactate) value for the flavin domain increases about 2-fold at moderate concentrations of glycerol. The data are consistent with a minimal chemical mechanism in which the lactate hydroxyl proton is not in flight in the transition state for C-H bond cleavage and there is an internal equilibrium involving the lactate-bound enzyme prior to C-H bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium.

Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation.

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxylase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in tyrosine hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine hydroxylase identified three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in tyrosine hydroxylase at the position corresponding to position 379 of phenylalanine hydroxylase is critical for dihydroxyphenylalanine formation. The relative specificity of tyrosine hydroxylase for phenylalanine versus tyrosine, as measured by the (V/K(phe))/(V/K(tyr)) value, increased by 80000-fold in the D425V enzyme. However, mutation of the corresponding valine 379 of phenylalanine hydroxylase to aspartate was not sufficient to allow phenylalanine hydroxylase to form dihydroxyphenylalanine at rates comparable to that of tyrosine hydroxylase. The double mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, showing a 3000-fold decrease in the (V/K(phe))/(V/K(tyr)) value.

Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation.

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.

Phenylalanine residues in the active site of tyrosine hydroxylase: mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300.

Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding. Also based on the structure, Phe300 has been reported to be hydroxylated due to a naturally occurring posttranslational modification [Goodwill, K. E., Sabatier, C., and Stevens, R. C. (1998) Biochemistry 37, 13437-13445]. Mutants of tyrosine hydroxylase with alanine substituted for Phe300 or Phe309 have now been purified and characterized. The F309A protein possesses 40% less activity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the production of dihydropterin. The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPA and dihydropterin. The K(6-MPH4) value for F300A tyrosine hydroxylase is twice the wild-type value. These results are consistent with Phe309 having a role in maintaining the integrity of the active site, while Phe300 contributes less than 1 kcal/mol to binding tetrahydropterin. Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolated catalytic domain after incubation with a large excess of 7, 8-dihydropterin, DTT, and Fe(2+). The modification is not observed in the untreated catalytic domain or in the full-length protein, even in the presence of excess iron. These results establish that hydroxylation of Phe300 is an artifact of the crystallography conditions and is not relevant to catalysis.

Site-directed mutants of charged residues in the active site of tyrosine hydroxylase.

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.

Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine.

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.

Expression and characterization of the catalytic core of tryptophan hydroxylase.

Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4a-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.

Characterization of chimeric pterin-dependent hydroxylases: contributions of the regulatory domains of tyrosine and phenylalanine hydroxylase to substrate specificity.

Tyrosine and phenylalanine hydroxylases contain homologous catalytic domains and dissimilar regulatory domains. To determine the effects of the regulatory domains upon the substrate specificities, truncated and chimeric mutants of tyrosine and phenylalanine hydroxylase were constructed: Delta117PAH, the C-terminal 336 amino acid residues of phenylalanine hydroxylase; Delta155TYH, the C-terminal 343 amino acid residues of tyrosine hydroxylase; and 2 chimeric proteins, 1 containing the C-terminal 331 residues of phenylalanine hydroxylase and the N-terminal 168 residues of tyrosine hydroxylase, and a second containing the C-terminal 330 residues of tyrosine hydroxylase and the 122 N-terminal residues of phenylalanine hydroxylase. Steady-state kinetic parameters with tyrosine and phenylalanine as substrate and the need for pretreatment with phenylalanine for full activity were determined. The truncated proteins showed low binding specificity for either amino acid. Attachment of either regulatory domain greatly increased the specificity, but the specificity was determined by the catalytic domain in the chimeric proteins. All three proteins containing the catalytic domain of phenylalanine hydroxylase were unable to hydroxylate tyrosine. Only wild-type phenylalanine hydroxylase required pretreatment with phenylalanine for full activity with tetrahydrobiopterin as substrate.

Expression and characterization of the catalytic domain of human phenylalanine hydroxylase.

A truncated version of human phenylalanine hydroxylase which contains the carboxy terminal 336 amino acids was produced in Escherichia coli. It was purified by ammonium sulfate precipitation, Q-Sepharose chromatography, and hydroxyapatite chromatography. The K(m) values of the truncated enzyme for tetrahydropterin substrates are not different from those of the full-length enzyme, nor are the Vmax values. The KM value for phenylalanine is 2-fold lower for the truncate than for the full-length enzyme. The metal content of the enzyme is 0.27 mol Fe per mole enzyme subunit, and it is activated 2.3-fold by addition of ferrous ion to assays; it is not activated by addition of copper. The truncated enzyme shows no lag in activity when an assay is started with phenylalanine, while the full-length enzyme shows a marked lag.

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.

Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose. A series of N-terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin-binding site. The mutants lacking the first 32 or 68 amino acids bind to heparin-Sepharose. The mutant lacking 76 amino acids binds somewhat to heparin-Sepharose and the proteins lacking 88 or 128 do not bind at all. Therefore, an important segment of the heparin-binding site must be composed of the region from residues 76 to 90. All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild-type enzymes.

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.

Lysine241 of tyrosine hydroxylase is not required for binding of tetrahydrobiopterin substrate.

The lysine residues at positions 194 and 198 in phenylalanine hydroxylase have been shown to react with a photoaffinity label which is an analog of phenyltetrahydropterin (Gibbs, B. S., and Benkovic, S. J. (1991) Biochemistry 30, 6795-6802), in a manner suggesting that these lysine residues are involved in tetrahydrobiopterin binding. The related enzyme tyrosine hydroxylase has a lysine at position 241 which, given the 75% identity between its C-terminal 330 amino acids and those of phenylalanine hydroxylase, corresponds to lysine194 of phenylalanine hydroxylase. Site-directed mutagenesis was used to alter lysine241 of tyrosine hydroxylase to alanine. Steady-state kinetic parameters were measured for wild-type and K241A tyrosine hydroxylase. No kinetic parameter differed between the wild-type and K241A enzymes, including Vmax values, Michaelis constants for tetrahydrobiopterin, 6-methyl-tetrahydropterin, and tyrosine, and the inhibition constants for norepinephrine. These results show that lysine241 is not required for tetrahydrobiopterin binding to tyrosine hydroxylase.

Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.

Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.

Cloning and expression of the luxY gene from Vibrio fischeri strain Y-1 in Escherichia coli and complete amino acid sequence of the yellow fluorescent protein.

Vibrio fischeri strain Y-1 (ATCC 33715) emits light with a lambda max of 545 nm rather than the 485-nm emission typical of other strains of V. fischeri. The yellow emission is due to the interaction of the enzyme luciferase with a yellow fluorescent protein (YFP). On the basis of the N-terminal amino acid sequence of YFP, a mixed-sequence oligonucleotide probe was synthesized and used to isolate a 1.6-kbp HindIII fragment containing the first 208 bases of the gene that codes for YFP (luxY). Another synthetic oligonucleotide complementary to bases 167-184 of the YFP coding sequence was used to isolate a second (ca. 1.9 kbp) DNA fragment generated by digestion with both EcoRI and ClaI that contained the remainder of the luxY gene. The intact luxY gene, which encoded a 22,211-dalton polypeptide composed of 194 amino acid residues, was reconstructed from the two primary clones and is contained within a 765-bp SspI-XhoII fragment. Both strands of the entire luxY coding sequence were determined from the reconstructed gene, while the region surrounding the junction used in the reconstruction was also determined from the original partial clones. As with other genes that have been studied from V. fischeri, the luxY gene was unusually AT-rich. The sequence of luxY did not bear any apparent similarity to any of the sequences contained in the current GenBank database. Escherichia coli containing a plasmid with the luxY gene expresses a protein that reacts with antibody raised to authentic YFP.

Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.

Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5.

Interaction between luciferases from various species of bioluminescent bacteria and the yellow fluorescent protein of Vibrio fischeri strain Y-1.

The interaction of the Yellow Fluorescent Protein (YFP) from Vibrio fischeri strain Y-1 with luciferases from other bioluminescent bacterial strains have been studied. Addition of purified YFP to a Y-1 luciferase assay results in enhancement of the intensity of blue (484 nm) bioluminescence and a new peak in the emission spectrum at about 540 nm. The bimodal spectrum also resulted when the luciferase used in the reaction was isolated from the species V. fischeri ATCC 7744, V. fischeri Y-1, Photobacterium phosphoreum, and P. leiognathi, but not when the luciferase was from V. harveyi. Analysis of the degree of enhancement versus the amount of YFP added yielded a binding constant for YFP to luciferase (V. fischeri Y-1 and V. harveyi) of about 2.4-4.0 microM.

Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein.

A strain of luminous bacteria, Vibrio fischeri Y-1, emits yellow light rather than the blue-green emission typical of other luminous bacteria. The yellow emission has been postulated previously to result from energy transfer from an electronically excited species formed in the bacterial luciferase-catalyzed reaction to a secondary emitter protein, termed the yellow fluorescent protein (YFP). We report here the purification of YFP to homogeneity without loss of the chromophore. The protein was found to be a homodimer of Mr 22,000 subunits with one weakly bound FMN per subunit. The FMN-protein complex was stabilized by 10% (vol/vol) glycerol in the buffers, allowing purification of the active holo-YFP. The protein migrated as a single spot with an isoelectric point of approximately 6.5 on two-dimensional polyacrylamide gel electrophoresis and gave an N-terminal sequence of Met-Phe-Lys-Gly-Ile-Val-Glu-Gly-Ile-Gly-Ile-Ile-Glu-Lys-Ile. Addition of purified YFP to a reaction in which luciferase was supplied with FMNH2 (reduced FMN) by a NADH:FMN oxidoreductase resulted in a dramatic enhancement in the intensity of bioluminescence and an additional peak in the emission spectrum at about 534 nm. The resulting bimodal bioluminescence emission spectrum had peaks at 484 nm, apparently due to emission from the luciferase-flavin complex, and at 534 nm, corresponding to the fluorescence emission maximum of YFP. This bimodal spectrum closely matched the emission spectrum in vivo.