Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol.

Tetrachlorohydroquinone dehalogenase catalyzes the replacement of chlorine atoms on tetrachlorohydroquinone and trichlorohydroquinone with hydrogen atoms during the biodegradation of pentachlorophenol by Sphingomonas chlorophenolica. The sequence of the active site region of tetrachlorohydroquinone dehalogenase is very similar to those of the corresponding regions of maleylacetoacetate isomerases, enzymes that catalyze the glutathione-dependent isomerization of a cis double bond in maleylacetoacetate to the trans configuration during the catabolism of phenylalanine and tyrosine. Furthermore, tetrachlorohydroquinone dehalogenase catalyzes the isomerization of maleylacetone (an analogue of maleylacetoacetate) at a rate nearly comparable to that of a bona fide bacterial maleylacetoacetate isomerase. Since maleylacetoacetate isomerase is involved in a common and presumably ancient pathway for catabolism of tyrosine, while tetrachlorohydroquinone dehalogenase catalyzes a more specialized reaction, it is likely that tetrachlorohydroquinone dehalogenase arose from a maleylacetoacetate isomerase. The substrates and overall transformations involved in the dehalogenation and isomerization reactions are strikingly different. This enzyme provides a remarkable example of Nature's ability to recruit an enzyme with a useful structural scaffold and elaborate upon its basic catalytic capabilities to generate a catalyst for a newly needed reaction.

The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases.

Kinetic parameters and primary deuterium isotope effects have been determined for wild-type dihydropteridine reductase (EC 1.6.99.7) and the Ala133Ser, Lys150Gln, Tyr146His, Tyr146Phe single, and Tyr146Phe/Ala133Ser and Tyr146Phe/Lys150Gln double mutant enzyme forms using the natural substrate, quinonoid (6R)-l-erythro-dihydrobiopterin (qBH2) and an alternate substrate, quinonoid 6,7-dimethyldihydropteridine (q-6,7-diMePtH2). Mutation at either Tyr146 or Lys150 resulted in pronounced changes in kinetic parameters and isotope effects for both pterin substrates, confirming a critical role for these residues in enzyme-mediated hydride transfer. By contrast, the Ala133Ser mutant was practically indistinguishable from wild-type enzyme. The changes observed, however, were quite different for the two pterin substrates. Thus, kcat for q-6,7-diMePtH2 decreased across the series of mutants from a value of 150 s-1 for wild-type enzyme to essentially zero activity for the Tyr146Phe/Lys150Gln double mutant. Conversely, kcat for qBH2 increased 3-11-fold across the same series of mutants from the wild-type value of 23 s-1. For both pterin substrates, the Km (KPt) increased several orders of magnitude upon mutation of Tyr146 or Lys150, with the greater relative increase using qBH2. Significant primary deuterium isotope effects on kcat (Dkcat) and kcat/KPt (D(kcat/KPt)) observed for the Tyr146 and Lys150 mutants varied depending on the pterin substrate used and ranged up to a maximum value of 5.5-6. For qBH2, where Dkcat < Dkcat/KPt was consistently observed, the rate determining step is ascribed to release of the tetrahydropterin product. For q-6,7-diMePtH2, where in all cases Dkcat = Dkcat/KPt, catalysis is probably limited by an isomerization step occurring prior to hydride transfer. Modeling studies in which qBH2 was docked into the binary E:NADH complex provide a structural rationale for the observed differences between the two pterin substrates. The natural substrate, qBH2, displays a higher affinity for the enzyme active site, presumably due to interaction of the dihydroxypropyl side chain of the substrate with a polar loop of residues containing Asn186, Ser189, and Met190. The location of this loop within the three-dimensional structure is consistent with putative substrate binding loops for other members of the short chain dehydrogenase/reductase (SDR) family, which includes dihydropteridine reductase.

Altered structural and mechanistic properties of mutant dihydropteridine reductases.

Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.

Structural and mechanistic characteristics of dihydropteridine reductase: a member of the Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases.

Dihydropteridine reductase (EC 1.6.99.7) is a member of the recently identified family of proteins known as short-chain dehydrogenases. When the x-ray structure of dihydropteridine reductase is correlated with conserved amino acid sequences characteristic of this enzyme class, two important common structural regions can be identified. One is close to the protein N terminus and serves as the cofactor binding site, while a second conserved feature makes up the inner surface of an alpha-helix in which a tyrosine side chain is positioned in close proximity to a lysine residue four residues downstream in the sequence. The main function of this Tyr-Lys couple may be to facilitate tyrosine hydroxyl group participation in proton transfer. Thus, it appears that there is a distinctive common mechanism for this group of short-chain or pyridine dinucleotide-dependent oxidoreductases that is different from their higher molecular weight counterparts.

Cyclotron production of NCA 99mTc and 99Mo. An alternative non-reactor supply source of instant 99mTc and 99Mo----99mTc generators.

The direct production of no-carrier-added (NCA) 6.02 h 99mTc and of 66 h 99mMo using proton beams of natural Mo targets was investigated. The major objective of this work was to evaluate the potential of utilizing high-intensity proton accelerators as a supply source of 99mTc and 99Mo for use in diagnostic nuclear medicine. The excitation functions for the production of the directly-made 99mTc via the 100Mo(p, 2n)99mTc (Q = -7.85 MeV) reaction, and of its parent 99Mo via the 100Mo(p, pn) 99Mo (Q = -8.30 MeV) and 100Mo(p, 2p)99mNb(15 s)----99Mo (Q = -11.14 MeV) reactions, were measured in the 68-8 MeV energy range. Single and cumulative yields for 99mTc and 99Mo, and for other Tc, Mo, Zr, Nb and Y radiocontaminants were also determined. The prospects of integrating the use of enriched 100Mo targets with high-intensity, dual beam, H- accelerators was analyzed. The potential of this combined method to replace or complement the current reactor-based supply sources of 99Mo----99mTc generators, is also discussed. Finally, a brief analysis is made on the potential use of this combined technology to support the anticipated expansion of nuclear medicine in developing nations.