A mechanistic investigation of the thiol-disulfide exchange step in the reductive dehalogenation catalyzed by tetrachlorohydroquinone dehalogenase

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachloro- and trichlorohydroquinone to give 2,6-dichlorohydroquinone in the pathway for degradation of pentachlorophenol by Sphingobium chlorophenolicum. Previous work has suggested that this enzyme may have originated from a glutathione-dependent double bond isomerase such as maleylacetoacetate isomerase or maleylpyruvate isomerase. While some of the elementary steps in these two reactions may be similar, the final step in the dehalogenation reaction, a thiol-disulfide exchange reaction that removes glutathione covalently bound to Cys13, certainly has no counterpart in the isomerization reaction. The thiol-disulfide exchange reaction does not appear to have been evolutionarily optimized. There is little specificity for the thiol; many thiols react at high rates. TCHQ dehalogenase binds the glutathione involved in the thiol-disulfide exchange reaction very poorly and does not alter its pK(a) in order to improve its nucleophilicity. Remarkably, single-turnover kinetic studies show that the enzyme catalyzes this step by approximately 10000-fold. This high reactivity requires an as yet unidentified protonated group in the active site.     

Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach

The pathway for degradation of the xenobiotic pesticide pentachlorophenol in Sphingomonas chlorophenolica probably evolved in the past few decades by the recruitment of enzymes from two other catabolic pathways. The first and third enzymes in the pathway, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, may have originated from enzymes in a pathway for degradation of a naturally occurring chlorinated phenol. The second enzyme, a reductive dehalogenase, may have evolved from a maleylacetoacetate isomerase normally involved in degradation of tyrosine. This apparently recently assembled pathway does not function very well: pentachlorophenol hydroxylase is quite slow, and tetrachlorohydroquinone dehalogenase is subject to severe substrate inhibition.     

cis-beta-acetylacrylate is a substrate for maleylacetoacetate cis-trans isomerase. Mechanistic implications

Maleylacetoacetate cis-trans isomerase together with coenzyme glutathione catalyzes cis-trans isomerization of cis-beta-acetylacrylate strongly suggesting that the isomerase directly catalyzes isomerization of the diketo forms in addition to the ketoenol forms of maleylacetone and maleylacetoacetate. The isomerase exhibits wider substrate acceptance than previously thought.     

Inhibition of glutathione S-transferase zeta and tyrosine metabolism by dichloroacetate: a potential unifying mechanism for its altered biotransformation and toxicity.

Dichloroacetate (DCA) inhibits its own metabolism and is converted to glyoxylate by glutathione S-transferase zeta (GSTz). GSTz is identical to maleylacetoacetate isomerase, an enzyme of tyrosine catabolism that converts maleylacetoacetate (MAA) to fumarylacetoacetate and maleylacetone (MA) to fumarylacetone. MAA and MA are alkylating agents. Rats treated with DCA for up to five days had markedly decreased hepatic GSTz activity and increased urinary excretion of MA. When dialyzed cytosol obtained from human liver was incubated with DCA, GSTz activity was unaffected. In contrast, DCA incubation inhibited enzyme activity in dialyzed hepatic cytosol from rats. Incubation of either rat or human hepatic cytosol with MA led to a dose dependent inhibition of GSTz. These data indicate that humans or rodents exposed to DCA may accumulate MA and/or MAA which inhibit(s) GSTz and, consequently, DCA biotransformation. Moreover, DCA-induced inhibition of tyrosine catabolism may account for the toxicity of this xenobiotic in humans and other species.     

The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution.

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone and trichlorohydroquinone during the biodegradation of the xenobiotic compound pentachlorophenol by Sphingobium chlorophenolicum. The mechanism of this transformation is of interest because it is unusual and difficult, and because aerobic microorganisms rarely catalyze reductive dehalogenation reactions. Tetrachlorohydroquinone dehalogenase is a member of the glutathione S-transferase superfamily. Many enzymes in this superfamily are capable of catalyzing nucleophilic aromatic substitution reactions. On the basis of this precedent, we have considered a mechanism for tetrachlorohydroquinone dehalogenase that involves a nucleophilic aromatic substitution reaction, either via an S(N)Ar mechanism or an S(RN)1-like mechanism, in the initial part of the reaction. Mechanistic studies were carried out with the wild type enzyme and with the C13S mutant enzyme, which catalyzes only the initial steps in the reaction. Three findings eliminate the possibility of a nucleophilic aromatic substitution reaction. First, the product of such a reaction, 2,3,5-trichloro-6-S-glutathionylhydroquinone, is not a kinetically competent intermediate. Second, the enzyme can carry out the reaction when the substrate is deprotonated at the active site. Nucleophilic aromatic substitution should not be possible when the substrate is negatively charged. Third, substantial normal solvent kinetic isotope effects on k(cat) and k(cat)/K(M,TriCHQ) are observed. Nonenzymatic and enzymatic nucleophilic S(N)Ar reactions typically show inverse solvent kinetic isotope effects.     

Gene Structure, Chromosomal Location, and Expression Pattern of Maleylacetoacetate Isomerase

The gene for maleylacetoacetate isomerase (MAAI) (EC 5.2.1.2) was the last gene in the mammalian phenylalanine/tyrosine catabolic pathway to be cloned. We have isolated the human and murine genes and determined their genomic structure. The human gene spans a genomic region of approximately 10 kb, has 9 exons ranging from 50 to 528 bp in size, and was mapped to 14q24.3-14q31.1 using fluorescence in situ hybridization. The complete catabolic pathway of phenylalanine/tyrosine is normally restricted to liver and kidney, but the maleylacetoacetate isomerase gene is expressed ubiquitously. This suggests a possible second role for the MAAI protein different from phenylalanine/tyrosine catabolism. We have searched for mutations in the maleylacetoacetate isomerase gene in four cases of unexplained severe liver failure in infancy with clinical similarities to hereditary tyrosinemia type I (pseudotyrosinemia). Several amino acid changes were identified, but all were found to retain MAAI activity and thus represent protein polymorphisms. We conclude that MAAI deficiency is not a common cause of the pseudotyrosinemic phenotype.     

Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity

Maleylacetoacetate isomerase (MAAI), a key enzyme in the metabolic degradation of phenylalanine and tyrosine, catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate. Deficiencies in enzymes along the degradation pathway lead to serious diseases including phenylketonuria, alkaptonuria, and the fatal disease, hereditary tyrosinemia type I. The structure of MAAI might prove useful in the design of inhibitors that could be used in the clinical management of the latter disease. Here we report the crystal structure of human MAAI at 1.9 A resolution in complex with glutathione and a sulfate ion which mimics substrate binding. The enzyme has previously been shown to belong to the zeta class of the glutathione S-transferase (GST) superfamily based on limited sequence similarity. The structure of MAAI shows that it does adopt the GST canonical fold but with a number of functionally important differences. The structure provides insights into the molecular bases of the remarkable array of different reactions the enzyme is capable of performing including isomerization, oxygenation, dehalogenation, peroxidation, and transferase activity.     

Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site

Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid. The 2-ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.     

Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism

A glutathione transferase (GST) similar to zeta GSTs in animals and fungi has been cloned from Arabidopsis thaliana using RT-PCR. The Arabidopsis zeta GST (AtGSTZ1) was expressed in Escherichia coli as his-tagged polypeptides, which associated together to form the 50-kDa AtGSTZ1-1 homodimer. Following purification, AtGSTZ1-1 was assayed for a range of activities and compared with other purified recombinant plant GSTs from the phi, tau, and theta classes. AtGSTZ1-1 differed from the other GSTs in showing no glutathione conjugating activity toward xenobiotics and no glutathione peroxidase activity toward organic hydroperoxides. Uniquely among the plant GSTs, AtGSTZ1-1 showed activity as a maleylacetone isomerase (MAI). This glutathione-dependent reaction is analogous to the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, which occurs in the course of tyrosine catabolism to acetoacetate and fumarate. Thus, rather than functioning as a conventional GST, AtGSTZ1-1 appears to be involved in tyrosine degradation. In addition to the MAI activity, the AtGSTZ1-1 also catalyzed the glutathione-dependent dehalogenation of dichloroacetic acid to glyoxylic acid. This latter activity was used to demonstrate the presence of functional AtGSTZ1-1 inplanta.     

A quasi-Laue neutron crystallographic study of d-xylose isomerase

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.     

Mechanism of the severe inhibition of tetrachlorohydroquinone dehalogenase by its aromatic substrates

Tetrachlorohydroquinone (TCHQ) dehalogenase catalyzes the conversion of TCHQ to 2,6-dichlorohydroquinone during degradation of pentachlorophenol by Sphingobium chlorophenolicum. TCHQ dehalogenase is a member of the glutathione S-transferase superfamily. Members of this superfamily typically catalyze nucleophilic attack of glutathione upon an electrophilic substrate to form a glutathione conjugate and contain a single glutathione binding site in each monomer of the typically dimeric enzyme. TCHQ dehalogenase, in contrast to most members of the superfamily, is a monomer and uses 2 equiv of glutathione to catalyze a more complex reaction. The first glutathione is involved in formation of a glutathione conjugate, while the second is involved in the final step of the reaction, a thiol-disulfide exchange reaction that regenerates the free enzyme and forms GSSG. TCHQ dehalogenase is severely inhibited by its aromatic substrates, TCHQ and trichlorohydroquinone (TriCHQ). TriCHQ acts as a noncompetitive inhibitor of the thiol-disulfide exchange reaction required to regenerate the free form of the enzyme. In addition, dissociation of the GSSG product is inhibited by TriCHQ. The thiol-disulfide exchange reaction is the rate-limiting step in the reductive dehalogenation reaction under physiological conditions.     

Maleylacetoacetate isomerase (MAAI/GSTZ)-deficient mice reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism

In mammals, the catabolic pathway of phenylalanine and tyrosine is found in liver (hepatocytes) and kidney (proximal tubular cells). There are well-described human diseases associated with deficiencies of all enzymes in this pathway except for maleylacetoacetate isomerase (MAAI), which converts maleylacetoacetate (MAA) to fumarylacetoacetate (FAA). MAAI is also known as glutathione transferase zeta (GSTZ1). Here, we describe the phenotype of mice with a targeted deletion of the MAAI (GSTZ1) gene. MAAI-deficient mice accumulated FAA and succinylacetone in urine but appeared otherwise healthy. This observation suggested that either accumulating MAA is not toxic or an alternate pathway for MAA metabolism exists. A complete redundancy of MAAI could be ruled out because substrate overload of the tyrosine catabolic pathway (administration of homogentisic acid, phenylalanine, or tyrosine) resulted in renal and hepatic damage. However, evidence for a partial bypass of MAAI activity was also found. Mice doubly mutant for MAAI and fumarylacetoacetate hydrolase (FAH) died rapidly on a normal diet, indicating that MAA could be isomerized to FAA in the absence of MAAI. Double mutants showed predominant renal injury, indicating that this organ is the primary target for the accumulated compound(s) resulting from MAAI deficiency. A glutathione-mediated isomerization of MAA to FAA independent of MAAI enzyme was demonstrated in vitro. This nonenzymatic bypass is likely responsible for the lack of a phenotype in nonstressed MAAI mutant mice.     

Reaction of triosephosphate isomerase with L-glyceraldehyde 3-phosphate and triose 1,2-enediol 3-phosphate

Triosephosphate isomerase catalyzes the isomerization and/or racemization reactions of L-glyceraldehyde 3-phosphate (LGAP), the enantiomer of the physiological substrate. The reaction is inhibited by the active site directed reagent glycidol phosphate. The amount of protonation product formation catalyzed by a fixed enzyme concentration is nearly independent of increasing steady-state concentrations of triose 1,2-enediol 3-phosphate caused by buffer catalysis of LGAP deprotonation. Therefore, enzymatic protonation of the enediol or enediolate, which could account for the observed enzymatic catalysis of LGAP isomerization and/or racemization, is at best a minor reaction. Instead LGAP reacts directly at the enzyme active site. Triosephosphate isomerase catalysis of the protonation of triose 1,2-enediol 3-phosphate was expected because of the strong evidence supporting an enediol reaction intermediate for the overall reaction catalyzed by isomerase. The most reasonable explanation for the failure to observe enzymatic protonation is that in solution the enediol undergoes beta elimination of phosphate (t 1/2 is estimated to be 10(-6) s) faster than it can diffuse to and form a complex with isomerase.     

Mechanism of enzymatic dehalogenation of pentachlorophenol by Arthrobacter sp. strain ATCC 33790.

Pentachlorophenol (PCP) dehalogenase from Arthrobacter sp. strain ATCC 33790 converts PCP to tetrachlorohydroquinone. In labeling experiments with H(2)18O or 18O2, only with H(2)18O was labeled product found. However, unlabeled tetrachlorohydroquinone became labeled after incubation with the enzyme in H(2)18O. Therefore, distinction between an oxygenolytic or a hydrolytic dehalogenation mechanism for the PCP dehalogenase is not possible.     

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.     

Plant carotene cis-trans isomerase CRTISO: a new member of the FAD(RED)-dependent flavoproteins catalyzing non-redox reactions

The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-α/β-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates. The reaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene β-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction.     

Enzymatic characterization of 5-methylthioribose 1-phosphate isomerase from Bacillus subtilis

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138 microM, and showed that the maximum velocity of the reaction was 20.4 micromol min(-1) (mg protein)(-1). The optimum reaction temperature and reaction pH were 35 degrees C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol(-1). The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.     

The 4-oxalocrotonate tautomerase family of enzymes: how nature makes new enzymes using a beta-alpha-beta structural motif

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers. The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy. Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base. In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies. Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink. Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50. 4-OT is also the title enzyme of the 4-OT family of enzymes. The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif. The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position. Characterization of representative members has uncovered mechanistic and structural diversity. A new activity, a trans-3-chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities. Two new structures have also been found, along with the 4-OT hexamer. The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer. Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity. Amplification of this activity could have produced the full-fledged dehalogenase. The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes.     

The reaction pathway of the isomerization of d-xylose catalyzed by the enzyme d-xylose isomerase: A theoretical study

Different pathways of the metal-induced isomerization of D-xylose to D-xylulose are investigated and compared in detail using energy minimization and molecular dynamics simulation. Two theoretical models are constructed for the reaction: in vacuum and in the enzyme D-xylose isomerase. The vacuum model is constructed based on the X-ray structure of the active site of D-xylose isomerase. It contains the atoms directly involved in the reaction and is studied using a semi-empirical molecular orbital method (PM3). The model in the enzyme includes the effects of the enzyme environment on the reaction using a combined quantum mechanical and molecular mechanical potential. For both models, the structures of the reactants, products, and intermediate complexes along the isomerization pathway are optimized. The effects of the position of the “catalytic Mg²⁺ ion” on the energies of the reactions are studied. The results indicate: 1) in vacuum, the isomerization reaction is favored when the catalytic metal cation is at site A, which is remote from the substrate; 2) in the enzyme, the catalytic metal cation, starting from site A, moves and stays at site B, which is close to the substrate; analysis of the charge redistribution of the active site during the catalytic process shows that the metal ion acts as a Lewis acid to polarize the substrate and catalyze the hydride shift; these results are consistent with previous experimental observations; and 3) Lys183 plays an important role in the isomerization reaction. The ϵ-NH₃⁺ group of its side chain can provide a proton to the carboxide ion of the substrate to form a hydroxyl group after the hydride shift step. This role of Lys183 has not been suggested before. Based on our calculations, we believe that this is a reasonable mechanism and consistent with site-directed mutation experiments. © 1997 Wiley-Liss Inc.     

4-Hydroxyphenylpyruvate dioxygenase

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an Fe(II)-dependent, non-heme oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. This reaction involves decarboxylation, substituent migration and aromatic oxygenation in a single catalytic cycle. HPPD is a member of the alpha-keto acid dependent oxygenases that typically require an alpha-keto acid (almost exclusively alpha-ketoglutarate) and molecular oxygen to either oxygenate or oxidize a third molecule. As an exception in this class of enzymes HPPD has only two substrates, does not use alpha-ketoglutarate, and incorporates both atoms of dioxygen into the aromatic product, homogentisate. The tertiary structure of the enzyme would suggest that its mechanism converged with that of other alpha-keto acid enzymes from an extradiol dioxygenase progenitor. The transformation catalyzed by HPPD has both agricultural and therapeutic significance. HPPD catalyzes the second step in the pathway for the catabolism of tyrosine, that is common to essentially all aerobic forms of life. In plants this pathway has an anabolic branch from homogentisate that forms essential isoprenoid redox cofactors such as plastoquinone and tocopherol. Naturally occurring multi-ketone molecules act as allelopathic agents by inhibiting HPPD and preventing the production of homogentisate and hence required redox cofactors. This has been the basis for the development of a range of very effective herbicides that are currently used commercially. In humans, deficiencies of specific enzymes of the tyrosine catabolism pathway give rise to a number of severe metabolic disorders. Interestingly, HPPD inhibitor/herbicide molecules act also as therapeutic agents for a number of debilitating and lethal inborn defects in tyrosine catabolism by preventing the accumulation of toxic metabolites.