Evidence for the mechanism of hydroxylation by 4-hydroxyphenylpyruvate dioxygenase and hydroxymandelate synthase from intermediate partitioning in active site variants.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.

Activation of α-keto acid-dependent dioxygenases: application of an {FeNO}7/{FeO2}8 methodology for characterizing the initial steps of O2 activation.

The α-keto acid-dependent dioxygenases are a major subgroup within the O(2)-activating mononuclear nonheme iron enzymes. For these enzymes, the resting ferrous, the substrate plus cofactor-bound ferrous, and the Fe(IV)═O states of the reaction have been well studied. The initial O(2)-binding and activation steps are experimentally inaccessible and thus are not well understood. In this study, NO is used as an O(2) analogue to probe the effects of α-keto acid binding in 4-hydroxyphenylpyruvate dioxygenase (HPPD). A combination of EPR, UV-vis absorption, magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies in conjunction with computational models is used to explore the HPPD-NO and HPPD-HPP-NO complexes. New spectroscopic features are present in the α-keto acid bound {FeNO}(7) site that reflect the strong donor interaction of the α-keto acid with the Fe. This promotes the transfer of charge from the Fe to NO. The calculations are extended to the O(2) reaction coordinate where the strong donation associated with the bound α-keto acid promotes formation of a new, S = 1 bridged Fe(IV)-peroxy species. These studies provide insight into the effects of a strong donor ligand on O(2) binding and activation by Fe(II) in the α-keto acid-dependent dioxygenases and are likely relevant to other subgroups of the O(2) activating nonheme ferrous enzymes.

Product analysis and inhibition studies of a causative Asn to Ser variant of 4-hydroxyphenylpyruvate dioxygenase suggest a simple route to the treatment of Hawkinsinuria.

Hawkinsinuria is a severe inherited condition that has a significant impact on the health of infants. The disease manifests as metabolic acidosis that significantly slows the growth rate and induces persistent diarrhea and vomiting. Though other causes may exist, an autosomal dominant mutation that alters codon 241 of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene from encoding an asparagine to encoding a serine gives rise to the symptoms of the disease. The observed pattern of dominance of this mutation belies the paucity of reports of this disease in the literature and suggests that it may be rarely diagnosed. Diagnosis is based on the presence of 2-amino-3-{[2-(carboxymethyl)-2,5-dihydroxy-1-cyclohex-3-enyl]sulfanyl}propanoic acid (hawkinsin) in the urine. We have made the structurally equivalent mutation in the Streptomyces avermitilis (N245S) and rat (N241S) genes and shown that in both cases the N to S variant enzyme forms quinolacetic acid in place of the native product 2,5-dihydroxyphenylacetic acid (homogentisate). Importantly, the variant enzyme is highly active, establishing the basis for dominant pedigree pattern. Quinolacetic acid reacts readily by Michael addition with cellular thiols to form a two-electron oxidized form of hawkinsin. The N to S variants are also susceptible to inhibition by 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), a known inhibitor of wild-type HPPD. NTBC has been approved for use in the treatment of type I tyrosinemia and as such has an extensive history of use with infants. The N to S variant undergoes an apparent three-step binding mechanism with NTBC that forms with rate constants similar to those observed for the wild-type enzyme. Moreover, the extreme stability of the HPPD.NTBC complex suggests that NTBC would be a potent therapeutic for Hawkinisinuria that would alleviate the extreme frailty experienced in the early life period.

The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate.

The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH(2)CH(2)NH(3) (+)) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.

The structure of Apo R268A human aldose reductase: hinges and latches that control the kinetic mechanism.

Aldose reductase (AR) catalyzes the NADPH-dependent reduction of glucose and other sugars to their respective sugar alcohols. The NADP+/NADPH exchange is the rate-limiting step for this enzyme and contributes in varying degrees to the catalytic rates of other aldo-keto reductase superfamily enzymes. The mutation of Arg268 to alanine in human recombinant AR removes one of the ligands of the C2-phosphate of NADP+ and markedly reduces the interaction of the apoenzyme with the nucleotide. The crystal structure of human R268A apo-aldose reductase determined to a resolution of 2.1 A is described. The R268A mutant enzyme has similar kinetic parameters to the wild-type enzyme for aldehyde substrates, yet has greatly reduced affinity for the nucleotide substrate which greatly facilitates its crystallization in the apoenzyme form. The apo-structure shows that a high temperature factor loop (between residues 214 and 226) is displaced by as much as 17 A in a rigid body fashion about Gly213 and Ser226 in the absence of the nucleotide cofactor as compared to the wild-type holoenzyme structure. Several factors act to stabilize the NADPH-holding loop in either the 'open' or 'closed' conformations: (1) the presence and interactions of the nucleotide cofactor, (2) the residues surrounding the Gly213 and Ser226 hinges which form unique hydrogen bonds in the 'open' or 'closed' structure, and (3) the Trp219 "latch" residue which interacts with an arginine residue, Arg293, in the 'open' conformation or with a cysteine residue, Cys298, in the 'closed' conformation. Several mutations in and around the high temperature factor loop are examined to elucidate the role of the loop in the mechanism by which aldose reductase binds and releases its nucleotide substrate.

Structural and thermodynamic studies of simple aldose reductase-inhibitor complexes.

The competitive inhibition constants of series of inhibitors related to phenylacetic acid against both wild-type and the doubly mutanted C298A/W219Y aldose reductase have been measured. Van't Hoff analysis shows that these acids bind with an enthalpy near -6.8 kcal/mol derived from the electrostatic interactions, while the 100-fold differences in binding affinity appear to be largely due to entropic factors that result from differences in conformational freedom in the unbound state. These temperature studies also point out the difference between substrate and inhibitor binding. X-ray crystallographic analysis of a few of these inhibitor complexes both confirms the importance of a previously described anion binding site and reveals the hydrophobic nature of the primary binding site and its general plasticity. Based on these results, N-glycylthiosuccinimides were synthesized to demonstrate their potential in studies that probe distal binding sites. Reduced alpha-lipoic acid, an anti-oxidant and therapeutic for diabetic complications, was shown to bind aldose reductase with a binding constant of 1 microM.

Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide, NTBC.

Di- and triketone inhibitors of (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are both effective herbicides and therapeutics. The inhibitory activity is used to halt the production of lipophilic redox cofactors in plants and also in humans to prevent accumulation of toxic metabolic byproducts that arise from specific inborn defects of tyrosine catabolism. The three-dimensional structure of the Fe(II) form of HPPD from Streptomyces avermitilis in complex with the inhibitor 2-[2-nitro-4-(triflouromethyl)benzoyl]-1,3-cyclohexanedione (NTBC) has been determined at a resolution of 2.5 A. NTBC coordinates to the active site metal ion, located at the bottom of a wide solvent-accessible cavity in the C-terminal domain of the protein. The iron is liganded in a predominantly five-coordinate, distorted square-pyramidal arrangement in which Glu349, His187, and His270 are protein-derived ligands and two other ligands are from the 5' and 7' oxygens of NTBC. There is a low-occupancy water molecule in the sixth coordination site in one of the protomers. The distance to His270 is unusually long at 2.5 A, and its orientation is somewhat distorted from ideal ligand geometry to within 2.8 A of the inhibitor nitro group. In contrast to the tetrameric quartenary structure observed for HPPD from other bacterial sources, the asymmetric unit is composed of two weakly associated protomers with a buried surface area of 1266 A(2) and a total of 12 hydrogen-bonding and no electrostatic interactions. The overall tertiary structure is similar to that of HPPD from Pseudomonas fluorescens (Serre et al., (1999) Structure 7, 977-988), although the position of the C-terminal alpha-helix is dramatically shifted. This C-terminal alpha-helix provides Phe364, which in combination with Phe336 sandwiches the phenyl ring of the bound NTBC; no other significant hydrogen-bonding or charge-pairing interactions are observed. Moreover, the structure reveals that, with the exception of Val189, NTBC makes contacts to only fully conserved amino acids. The combination of bidentate metal-ion coordination and pi-stacked aromatic rings is suggestive of a binding mode for the substrate and/or a transition state, which may be the origin of the exceedingly high affinity these inhibitors have for HPPD.