Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease.

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.

Inactivation of tyrosine phenol-lyase by Pictet-Spengler reaction and alleviation by T15A mutation on intertwined N-terminal arm.

Citrobacter freundiil-tyrosine phenol-lyase (TPL) was inactivated by a Pictet-Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-L-alanine (L-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards L-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with L-DOPA was detected at 45 and 40 degrees C, respectively, which was 15 degrees C lower than when using L-tyrosine as the substrate. The half-lives at 45 degrees C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mM L-DOPA. On treatment with excess pyridoxal-5'-phosphate (PLP), the L-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet-Spengler condensation with L-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (K(m,PLP)) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 degrees C, implying that the loss of the cofactor with the Pictet-Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.

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.

The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity.

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Site-directed mutagenesis of His343-->Ala in Citrobacter freundii tyrosine phenol-lyase. Effects on the kinetic mechanism and rate-determining step.

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R.L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195--4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha, beta-elimination (except S-ethyl-L-cysteine) exhibited lower kcat (10-30%) and kcat/Km (1-10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for L-phenylalanine and L-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-L-tyrosine with wild-type tyrosine phenol-lyase is identical to that of L-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333-7338]. The pH profile of kcat/Km for this reaction exhibits two pKa values with an average of 7.7 +/- 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 +/- 0.3. However, kcat for 3-fluoro-L-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on kcat and kcat/Km for 3-fluoro-L-[alpha-2H]-tyrosine are independent of pH and equal to 3.9 +/- 0.2 and 2.2 +/- 0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 +/- 0.2 and 3.8 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations.

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.

Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity.

L-Amino acids are competitive inhibitors of tyrosine phenol-lyase from Citrobacter intermedius. For non-branched amino acids the correlation exists between -RTlnKi and side-chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This suggests the presence of an electrophilic group in the active site which interacts with the terminal carboxylic group of aspartic or glutamic acids. Tyramine, beta-phenylethylamine and tryptamine do not display detectable inhibition. The esters and amides of aromatic L-amino acids, D-phenylalanine and D-tryptophan are competitive inhibitors. The enzymatic isotope exchange of the alpha-proton in 2H2O was observed only in the case of L-amino acids. For L-phenylalanine and L-tryptophan it was shown to proceed with complete retention of configuration. The substrate specificity of tyrosine phenol-lyase is controlled during the stage of phenol elimination. The OH group in the para position of the ring is necessary for this stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When all the requirements of substrate specificity are fulfilled (L-tyrosine, 3-fluoro-L-tyrosine), the 'key' phenol-elimination step is not the rate-limiting one, the reaction velocity being determined by the preceding alpha-proton abstraction.

[Interaction of tyrosine-phenol-lyase from Citrobacter intermedius with amino acids and their derivatives: factors determining the effectiveness of binding]. / Vzaimodeistvie tirozin-fenol-liazy iz Citrobacter intermedius s aminokislotami i ikh proizvodnymi: faktory, opredeliaiushchie éffektivnost' sviazyvaniia.

The inhibition by L-amino acids and their derivatives of tyrosine phenol-lyase is investigated. Tyramine, alpha-phenylethylamine and tryptamine have no detectable inhibition effect and hence are weakly bonded by an active site. The aromatic amino acid amides are competitive inhibitors but do not manifest an enzymatic isotope exchange of alpha-proton in D2O. Free amino acids however are competitive inhibitors and in the majority of cases exchange alpha-proton. The presence of COOH-group is therefore an important feature which determines the binding efficiency and causes the "active" conformation of the amino acid-PLP complex labelising alpha-proton. In the absence of functional and bulky groups in the amino acid side chain the hydrophobicity is found to be the main factor determining the binding efficiency. For these amino acids a correlation exists between-RTlnKi and side chain hydrophobicity. The amino acids bearing the bulky groups, i. e. valine, leucine and isoleucine have reduced binding efficiency. Lysine and arginine bearing positively charged functional groups possess no inhibition effect. Aspartic and glutamic acids are anomalously strong inhibitors taking into consideration low hydrophobicity of their side chains. One can assume that the electrophilic group able to interact with the terminal COO- -group of aspartic and glutamic acids is located in the active site of tyrosine phenollyase.

Transamination catalysed by tyrosine phenol-lyase from Citrobacter intermedius.

The interactions of tyrosine phenol-lyase with its substrates: L-tyrosine and L-serine, and the competitive inhibitors: L-alanine, L-phenylalanine, L-m-tyrosine, were studied. It was demonstrated that the enzyme catalyzed a half-transamination reaction between substrates or inhibitors and the protein-bound pyridoxal phosphate. The products of this side-reaction, pyridoxamine phosphate and the respective keto acids, were identified. The kinetic parameters were determined for beta-elimination of L-tyrosine and of L-serine, and for the transamination of L-serine and the inhibitors used. The transfer of the amino group to the coenzyme takes place in the direction from amino acid to pyridoxal phosphate, but not in the opposite direction, i.e. the transamination is irreversible.

[Substrate specificity of tyrosine-phenol-lyase. Electron and steric control at the stage of aromatic moiety elimination]. / Substratnaia spetsifichnost' tirozin-fenol-liazy. Elektronnyi i stericheskii kontrol' na stadii otshchepleniia aromaticheskogo fragmenta.

We have investigated the electronic and steric effects of substituents in the aromatic moiety of the substrate on the two principal stages of the reaction catalyzed by tyrosine-phenol-lyase. The substrate specificity of the enzyme is controlled during the stage of elimination of the aromatic ring. The process may be formally considered as an electrophilic substitution in the aromatic nucleus and includes tautomerization of the phenol group into cyclohexadienone and subsequent beta-elimination with regeneration of aromaticity in the leaving group. The OH-group in the rho-position of the ring is the first necessary condition for the stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When the requirements of substrate specificity are fulfilled (L-tyrosine, 3-F-L-tyrosine) the "key" stage of elimination of phenol moiety is not the rate-limiting one, the velocity of the reaction being determined by the preceding stage of alpha-proton abstraction.

[Effect of monovalent cations on the catalytic and spectral properties of tyrosine-phenol-lyase from Citrobacter intermedius]. / Vliianie monovalentnykh kationov na kataliticheskie i spektral'nye svoistva tirozin-fenol-liazy iz Citrobacter intermedius.

The action of monovalent cations Li+, Na+, K+, Rb+, Cs+, NH4+ on catalytic and physico-chemical properties of bacterial tyrosine--phenol-lyase was investigated. It was shown that K+, Rb+, Cs+, NH4+ were the noncompetitive activators of the enzyme, Na+ was an inhibitor, Li+ did not influence the catalytic activity. The values of KA and Vmax were determined for the activators in the reaction of alpha, beta-elimination of L-tyrosine. Monovalent cations affect the absorption and CD spectra of the enzyme and its complex with the quasi-substrate--L-alanine. It was suggested that an activation of tyrosine phenollyase by monovalent cations was connected with the increase of the active protonated form of the holoenzyme (lambda max 420 mm) induced by the cations-activators.

Properties of stearylamine liposomes containing tyrosine phenol-lyase.

The activity of tyrosine phenol-lyase, a chemotherapeutic enzyme with a dissociable pyridoxal phosphate cofactor, was studied after incorporation into multilamellar positively charged liposomes. Tyrosine phenol-lyase activity was assessed in the presence and absence of exogenous pyridoxal phosphate. A maximum of 75% total enzyme activity was associated with liposomes when prepared from a molar lipid ratio of egg lecithin, cholesterol, stearylamine (7 : 2 : 1, w/w). The total tyrosine phenol-lyase activity was comprised of 25% membrane-associated enzyme and 50% encapsulated enzyme. Encapsulation increased the stability of the enzyme under the in vitro conditions of cold storage at 4 degrees C for 3 weeks and under elevated temperatures up to 61 degrees C. Liposomal encapsulation afforded little protection against trypsin and no protection against whole mouse plasma in vitro. Heat-treated plasma (100 degrees C for 1 h) had little effect on the activity of free and encapsulated tyrosine phenol-lyase. These results indicated that whole plasma contained a heat-labile factor(s) which destroyed both the liposomal and free tyrosine phenol-lyase activity. Plasma clearance after intraperitoneal injection of tyrosine phenol-lyase in B6D2F1 female mice was retarded by liposomal encapsulation, particularly when the animals were pre-treated with empty liposomes; however, only a small proportion of free and liposomal tyrosine phenol-lyase was absorbed. The free enzyme rapidly lost holoenzyme activity after absorption but the liposomes maintained holoenzyme activity. Even though liposomes preserved holo-tyrosine phenol-lyase activity, the holoenzyme was not present in sufficient concentration to sustain a reduced plasma tyrosine level.