Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase

Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate. These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates. We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations. Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E. coli Trpase, are essential for activity with both substrates. Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates. Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies. T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC. T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr. F448H TPL also has very low activity with L-Tyr. None of the mutant TPLs has any detectable activity with L-Trp as substrate. H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates. Thus, additional residues remote from the active site may be needed for substrate specificity. Both Trpase and TPL may react by a rare S(E)2-type mechanism.     

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

The whole cell tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) activity of Erwinia herbicola (ATCC 21434) was microen-capsulated. We studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 degrees C to 32 degrees C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 degrees C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42 degrees C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 +/- 100 mum, mean +/- std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of-the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K(M) of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K(M) for phenol is 0.5 mM. The apparent K(M) values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme. (c) 1995 John Wiley & Sons, Inc.     

Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine

Incorporation of 3-fluorotyrosine and site-specific mutagenesis has been utilized with Fourier transform infrared (FTIR) spectroscopy and x-ray crystallography to elucidate active-site structure and the role of an active-site residue Tyr34 in human manganese superoxide dismutase (MnSOD). Calculated harmonic frequencies at the B3LYP/6-31G** level of theory for L-tyrosine and its 3-fluorine substituted analog are compared to experimental frequencies for vibrational mode assignments. Each of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD was replaced with 3-fluorotyrosine. The crystal structures of the unfluorinated and fluorinated wild-type MnSOD are nearly superimposable with the root mean-square deviation for 198 alpha-carbon atoms at 0.3 A. The FTIR data show distinct vibrational modes arising from 3-fluorotyrosine in MnSOD. Comparison of spectra for wild-type and Y34F MnSOD showed that the phenolic hydroxyl of Tyr34 is hydrogen bonded, acting as a proton donor in the active site. Comparison with crystal structures demonstrates that the hydroxyl of Tyr34 is a hydrogen bond donor to an adjacent water molecule; this confirms the participation of Tyr34 in a network of residues and water molecules that extends from the active site to the adjacent subunit.     

The effect of phenol on growth and induction of tyrosine phenol lyase of escherichia intermedia and Erwinia herbicola

Summary L-Tyrosine phenol lyase (TPL) biosynthesis is induced by the presence of L-tyrosine in the culture medium of E. intermedia and E. herbicola. Cell growth and induction of TPL were strongly reduced by the presence of added phenol in the culture media of the two bacteria. Adsorption of phenol on an ion exchange resin reversed nearly completely the observed inhibition.     

Inhibition of tyrosine phenol-lyase from Citrobacter freundii by 2-azatyrosine and 3-azatyrosine.

The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined. 2-Aza-DL-tyrosine and 3-aza-DL-tyrosine were synthesized by standard methods of amino acid synthesis, while the L-isomers were prepared from 3-hydroxypyridine and 2-hydroxypyridine, respectively, with TPL (Watkins, E. B., and Phillips, R. S. (2001) Bioorg. Med. Chem. Lett. 11, 2099-2100). 3-Azatyrosine was examined as a potential transition state analogue inhibitor of TPL. Both compounds were found to be competitive inhibitors of TPL, with K(i) values of 3.4 mM and 135 microM for 3- and 2-aza-L-tyrosine, respectively. Thus, 3-azatyrosine does not act as a transition state analogue, possibly due to the lack of tetrahedral geometry at C-1. However, 2-aza-L-tyrosine is the most potent competitive inhibitor of TPL found to date. The K(i) value of 2-aza-L-tyrosine is half that of 2-aza-DL-tyrosine, indicating that the D-enantiomer is inactive as an inhibitor. Neither azatyrosine isomer was shown to be a substrate for beta-elimination, based on coupled assays with lactate dehydrogenase and on HPLC measurements. Both isomers of azatyrosine form equilibrium mixtures of external aldimine and quinonoid intermediates when they bind to TPL. However, 2-azatyrosine reacts about 10-fold faster to form a quinonoid intermediate than does 3-azatyrosine. Since 2-azatyrosine is in the zwitterion or phenolate ion form at all the pH values examined, the strong binding of this compound suggests that L-tyrosine may be bound to the active site of TPL as the phenolate anion.     

Isolation and properties of tyrosine phenol-lyase from Citrobacter intermedius

A method for preparation of homogeneous tyrosine phenol lyase (EC 4.199.2) from Citrobacter intermedius has been developed. The cells were cultivated in the media with a view to obtain a cell culture with a high activity of tyrosine phenol lyase. The isoelectric point for the enzyme lies at pH 4.9. Tyrosine phenol lyase is strictly stereospecific: it catalyzes the formation of pyruvate only from L-tyrosine, but not from D-tyrosine. Kinetic studies showed that K+ and NH4+ cations are non-competitive activators of the enzyme (Ka = 3.57 X 10(-3) and 1.34 X 10(-4) M, respectively).     

Cloning and expression of the Erwinia herbicola tyrosine phenol-lyase gene in Escherichia coli.

The tyrosine phenol-lyase (TPL) gene of Erwinia herbicola was cloned and expressed in Escherichia coli, and the complete nucleotide sequence of the gene determined. The TPL gene comprises 1368 bp, encoding 456 amino acids which have 90% amino acid identity with TPL from Citrobacter freundii. After replacing the 5'-flanking region of the TPL gene with the E. coli lac promoter, TPL protein could be hyperproduced constitutively in E. coli without induction by L-tyrosine.     

Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway

The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2' '-position of UDP-3-O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus, a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k(cat)/K(M) and k(cat) for the deacetylation of the substrate UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK(1)) was fitted to 6.1 +/- 0.2 for k(cat) and 5.7 +/- 0.2 for k(cat)/K(M). The descending basic limb (pK(2)) was fitted to 8.0 +/- 0.2 for k(cat) and 8.4 +/- 0.2 for k(cat)/K(M). The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water.     

Phenol and lactone receptors in the distal sensilla of the Haller's organ in Ixodes ricinus ticks and their possible role in host perception

Extract of steer wool odor was found to excite olfactory receptor(s) in a wall-pore olfactory sensillum on the distal knoll of the Haller's organ. Three active volatile compounds were revealed in this odor by gas chromatography. Electrophysiological experiments revealed two types of receptors (sensory neurons) within the sensilla examined. One type of receptor responded only to phenolic derivatives, such as o-chlorophenol, o-bromophenol, o-methylphenol, 2,6-dichlorophenol, 2,6-dibromophenol, 2,4,6-trichlorophenol, but not to o-nithrophenol, p-methylphenol, 2,5-dichlorophenol, 3,5-dichlorophenol, 2,6-dinithrophenol, 2,6-dimethylphenol, and pentachlorophenol. The other type of receptor responded only to gamma-valerolactone. It is assumed that these cells play an important role in perception of a host from long distances (10-15 m), which is typical of Ixodes ricinus ticks.     

Development of a multienzyme reactor for dopamine synthesis: I. enzymology and kinetics

The enzymology and kinetics of tyrosine phenol lyase (TPL) from Erwinia herbicola, and tyrosine decarboxylase (TDC) from Streptococcus faecalis have been investigated for potential use in a coimmobilized multienzyme biocatalytic system for the production of dopamine. In this multienzyme biotransformation using whole cells optimized for each of the respective enzymes, TPL catalyzes the production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) from catechol, pyruvate, and ammonium, and this is subsequently decarboxylated by TDC to produce dopamine. Performing the reactions simultaneously, thereby removing L-dopa, is one option for overcoming the TPL equilibrium constraints. The enzymes have different optimal pH values, so the reaction kinetics at a compromise pH of 7.1, where both enzymes could be operated simultaneously, were investigated. For the concentration range investigated, TPL followed pseudo-first-order kinetics with respect to catechol, pyruvate, and ammonium. TDC exhibited significant product inhibition as well as inhibition by combinations of catechol and pyruvate.     

3-Fluorotyrosine as a Complementary Probe of Hemoglobin Structure and Dynamics: A (19) F-NMR Study of Synechococcus sp. PCC 7002 GlbN

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19) F-NMR spectroscopy for the characterization of GlbN. Assignment of (19) F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.     

Nucleotide binding to Na,K-ATPase: pK values of the groups affecting the high affinity site

Investigation of the ionic strength effect on the interactions between nucleotides (ATP and ADP) and Na,K-ATPase in a broad pH range was aimed at revealing pK values of the charged groups of the interacting species. Ionic strength experiments suggested that an amino acid residue with a pK > 8.0 is part of the protein binding site. A combination of equilibrium and transient experiments at various pH values allowed for the characterization of the groups electrostatically involved in either the association process (kon) or the stability of the preformed complexes (koff). Two groups (pK1 = 6.7 and pK2 = 8.4) appear to be important for the proper organization of the binding site and, therefore, the association reaction. Moreover, deprotonation of the basic group completely precludes association. pH dependencies of the dissociation rate constants for ATP and ADP are very different. An increase in pH from 5 to 9.5 induces a 9-fold increase in koff for ATP, whereas koff for ADP decreases 4-fold between pH 5 and 8, and decreases further in the alkaline region. A comparison of the pH dependencies for koff for ATP and ADP suggests two effects: (1) at acidic pH, the value of the total negative charge of the nucleotide determines the tightness of binding; and (2) short-range interactions involving the terminal phosphate group are important for nucleotide dissociation from the site. The difference in the pH dependencies of koff for the nucleotides suggests the existence of positive charges in close proximity to Asp369, relieving the repulsion between the gamma-phosphate of ATP and Asp369.     

Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.     

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.     

Catalytic properties of extracts from tyrosine phenol lyase producing cells of Escherichia intermedia: Multienzyme complex instead of the single multisubstrate enzyme

Kinetic parameters for the formation of pyruvate from L-tyrosine catalysed by the cell extract of Escherichia intermedia A-21 differ markedly from the parameters of crystalline tyrosine phenol lyase taken from the literature. The substrate specificity of the enzyme in the cell extract was also found to be different from that in the crystalline state. The cell extracts did not show any activity with respect to D-tyrosine, while the reactions with L-and D-enantiomers of serine were brought about mainly by active sites which differ kinetically from the active site responsible for the main reaction. The ratio of activities with respect to L-tyrosine, L-serine and D-serine varied widely depending on the composition of the medium on which the cells had been grown. The high activity of the preparation with respect to L-tyrosine is not a sufficient condition for successful tyrosine synthesis from dl-serine. High activities towards serine enantiomers are also necessary.     

Mutations in Salmonella typhimurium and inactivation of Bacillus subtilis transforming DNA induced by phenylhydroxylamine derivatives

Phenylhydroxylamine (PHA) and its derivatives such as monomethyl (2-Me, 3-Me, 4-Me) and dimethyl (2,3-diMe, 2,4-diMe, 2,5-diMe, 2,6-diMe, 3,4-diMe, 3,5-diMe) were tested for their mutagenicity and for their inducing ability to inactivate transforming DNA. All these compounds except PHA and 3,5-diMePHA were found to be mutagenic in Salmonella typhimurium TA100 even in the absence of S9 mix, and their mutagenic potency was in the order: 2,6-diMe- greater than 2,4-diMe- = 3,4-diMe- greater than 4-Me- greater than 2,3-diMe- = 2,5-diMe- greater than 2-Me- = 3-MePHA. Besides mutagenicities, all the PHA derivatives except 2,6-diMePHA caused severe reductions in the activity of Bacillus subtilis transforming DNA. To establish the structure-activity relationship, we examined the correlation between these activities and the stabilities of the PHA derivatives, and the results indicated that the more chemically unstable the PHA derivatives were, the more active they were with respect to the mutations and to the inactivation of the transforming DNA. The mutagenic activity of 2,6-diMePHA was the sole exception, because it was most stable, but its induced mutation frequency was highest. From these results, we suggest that all the PHA derivatives, except 2,6-diMePHA, cause DNA damage through the generation of active molecular species, such as nitrenium ions, without any enzymatic activation, while 2,6-diMePHA requires further metabolic activation by bacterial enzymes to stimulate mutagenesis.     

Chemical influences on the specificity of tyrosine phosphorylation

Biological tyrosine phosphorylation has become an extensively studied reaction. Little, however, is known of the chemistry involved. The acetylation of the tyrosyl phenolic hydroxyl group by N-acetylimidazole was studied as a model acylation reaction over the pH range 7.5-9.5. The reactivities of tyrosine and 3-fluorotyrosine were compared. The ratio of reactivities, kappa F-Tyr/kappa Tyr, decreases with increasing pH. Extrapolation to the state in which equivalent concentrations of the two derivatives exist indicates that, consistent with Br?nsted theory, tyrosine is 17 times more reactive than fluorotyrosine. No reactivity was observed with tetrafluorotyrosine, 3-nitrotyrosine, or 3,5-dinitrotyrosine. A peptide containing fluorotyrosine was synthesized and compared with the tyrosine-containing peptide as a substrate for the insulin receptor/tyrosine kinase. In both the presence and absence of insulin, the tyrosine peptide was phosphorylated with higher Vm and Km values than the fluorotyrosine peptide was. These results suggest that ionization of the tyrosyl hydroxyl group has an effect on both the chemical and enzymatic reactivities of the tyrosyl residue in acylation reactions. A model is suggested in which deprotonation of the acceptor occurs upon binding of the substrate to the kinase and implicates a role for the substrate site microenvironment in defining substrate specificity.     

Study of penicillin amidase from E. coli. pH-dependence of kinetic parameters of enzymatic hydrolysis of benzylpenicillin]

The authors studies pH-dependencies of the kinetic parameters (Vm, KM, Vm/KM) and constants of competitive inhibition by phenylacetic acid of penicillinamidase-catalyzed hydrolysis of benzylpenicillin. The experimental data are in agreement with the assumption according to which there are 3 equilibrium ionogenic forms of the enzyme and enzyme-substrate (or enzyme-inhibitor) complexes, i.e. acidic, neutral and alkaline, the neutral form being the only active form of the Michaelis complex. Values of pK in the ionogenic groups controlling interconversions of both the free enzyme (pK1 6.1 and pK2 7.6) and of the enzyme-substrate complex (pKa 6.1 and pK2 10.2 or the enzyzme-inhibitor complex (pK'1 6.1 and pK'2 9.5) were determined. From this and the previously published results it was concluded that the group with pK 6.1 was involved in the catalysis and the group with pK 10.2 in the maintenance of the active conformation of the active centre of penicillinamidase. The ionogenic group with pK 7.6 was apparently involved in the enzyme-substrate binding.     

Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the first step in the racemization of proline catalyzed by proline racemase

Rate constants for the hydrolysis of L-proline methyl ester to form proline and methanol in D(2)O buffered at neutral pD and 25 degrees C and the deuterium enrichment of the proline product determined by electrospray ionization mass spectrometry are reported. The data give k(DO) = 5.3 +/- 0.5 M(-1) s(-1) as the second-order rate constant for carbon deprotonation of N-protonated proline methyl ester by deuterioxide ion in D(2)O at 25 degrees C and I = 1.0 (KCl). The data provide good estimates of carbon acidities of pK(a) = 21 for N-protonated proline methyl ester and pK(a) = 29 for proline zwitterion in water and of the second-order rate constant k(HO) = 4.5 x 10(-5) M(-1) s(-1) for carbon deprotonation of proline zwitterion by hydroxide ion at 25 degrees C. There is no detectable acceleration of the deprotonation of N-protonated proline methyl ester by the Br?nsted base 3-quinuclidinone in water, and it is not clear that such Br?nsted catalysis would make a significant contribution to the rate acceleration for deprotonation of bound proline at proline racemase. A comparison of the first-order rate constants k(HO)[HO(-)] = 4.5 x 10(-11) s(-1) for deprotonation of free proline zwitterion in water at pH 8 and k(cat) = 2600 s(-1) for deprotonation of proline bound to the active site of proline racemase at pH 8 shows that the enzymatic rate acceleration for proline racemase is ca. 10(13)-fold. This corresponds to a 19 kcal/mol stabilization of the transition state for deprotonation of the enzyme-bound carbon acid substrate by interaction with the protein catalyst. It is suggested that (1) much of the rate acceleration of the enzymatic over the nonenzymatic reaction in water may result from transfer of the substrate proline zwitterion from the polar solvent water to a nonpolar enzyme active site and (2) the use of thiol anions rather than oxygen anions as Br?nsted bases at this putative nonpolar enzyme active site may be favored, because of the smaller energetic price for desolvation of thiol anions than for desolvation of the more strongly solvated oxygen anions.