Jeremiah W. Hanes

Incorporation and Replication of 8-Oxo-deoxyguanosine by the Human Mitochondrial DNA Polymerase

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 μm-1 s-1, a value ∼10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 μm-1 s-1, a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.

Pyridoxal phosphate: biosynthesis and catabolism

Vitamin B(6) is an essential cofactor that participates in a large number of biochemical reactions. Pyridoxal phosphate is biosynthesized de novo by two different pathways (the DXP dependent pathway and the R5P pathway) and can also be salvaged from the environment. It is one of the few cofactors whose catabolic pathway has been comprehensively characterized. It is also known to function as a singlet oxygen scavenger and has protective effects against oxidative stress in fungi. Enzymes utilizing vitamin B(6) are important targets for therapeutic agents. This review provides a concise overview of the mechanistic enzymology of vitamin B(6) biosynthesis and catabolism. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase.

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3′-azido-2′,3′-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5′-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s−1) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s−1, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/Km (7 × 10−6 μ M−1 s−1). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5′-triphosphate (8-oxo-dGTP) show this same phenomena.

Cofactor biosynthesis – still yielding fascinating new biological chemistry

This mini review covers recent advances in the mechanistic enzymology of cofactor biosynthesis.

Structural Similarities between Thiamin-Binding Protein and Thiaminase-I Suggest a Common Ancestor†,‡

ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.

Enzymatic Therapeutic Index of Acyclovir VIRAL VERSUS HUMAN POLYMERASE γ SPECIFICITY

We have examined the kinetics of incorporation of acyclovir triphosphate by the herpes simplex virus-1 DNA polymerase holoenzyme (Pol-UL42) and the human mitochondrial DNA polymerase using transient kinetic methods. For each enzyme, we compared the kinetic parameters for acyclovir to those governing incorporation of dGTP. The favorable ground state dissociation constant (6 μm) and rate of polymerization (10 s-1) afford efficient incorporation of acyclovir triphosphate by the Pol-UL42 enzyme. A discrimination factor of ∼50 favors dGTP over acyclovir triphosphate, mostly due to a faster maximum rate of dGTP incorporation. Once incorporated, acyclovir is removed with a half-life of ∼1 h in the presence of a normal concentration of deoxynucleoside triphosphates, leading to a high toxicity index (16,000) toward viral replication. To assess the potential for toxicity toward the host we examined the incorporation and removal of acyclovir triphosphate by the human mitochondrial DNA polymerase. These results suggest moderate inhibition of mitochondrial DNA replication defining a toxicity index of 380. This value is much higher than the value of 1.5 determined for tenofovir, another acyclic nucleoside analog. The enzymatic therapeutic index is only 42 in favoring inhibition of the viral polymerase over polymerase γ, whereas that for tenofovir is greater than 1,200. Mitochondrial toxicity is relatively low because acyclovir is activated only in infected cells by the promiscuous viral thymidine kinase and otherwise, mitochondrial toxicity would accumulate during long term treatment.

Trapping of a chromophoric intermediate in the Pdx1-catalyzed biosynthesis of pyridoxal 5'-phosphate.

Pyridoxal 5’-phosphate (PLP, 1) is the biologically active form of vitamin B6, and is essential for performing the chemistry of primary metabolism in all varieties of life. The most common pathway for the biosynthesis of PLP involves only two enzymes, Pdx1 (SNZ) and Pdx2 (SNO). This biosynthetic activity has recently been reconstituted in vitro and was shown to use ribose-5-phosphate (R5P, 2), glutamine 3, and glyceraldehyde-3-phosphate (G3P, 4) as substrates (Scheme 1).

Exonuclease Removal of Dideoxycytidine (Zalcitabine) by the Human Mitochondrial DNA Polymerase

ABSTRACT The toxicity of nucleoside analogs used for the treatment of human immunodeficiency virus infection is due primarily to the inhibition of replication of the mitochondrial genome by the human mitochondrial DNA polymerase (Pol γ). The severity of clinically observed toxicity correlates with the kinetics of incorporation versus excision of each analog as quantified by a toxicity index, spanning over six orders of magnitude. Here we show that the rate of excision of dideoxycytidine (zalcitabine; ddC) was reduced fourfold (giving a half-life of ∼2.4 h) by the addition of a physiological concentration of deoxynucleoside triphosphates (dNTPs) due to the formation of a tight ternary enzyme-DNA-dNTP complex at the polymerase site. In addition, we provide a more accurate measurement of the rate of excision and show that the low rate of removal of ddCMP results from both the unfavorable transfer of the primer strand from the polymerase to the exonuclease site and the inefficient binding and/or hydrolysis at the exonuclease site. The analogs ddC, stavudine, and ddATP (a metabolite of didanosine) each bind more tightly at the polymerase site during incorporation than normal nucleotides, and this tight binding contributes to slower excision by the proofreading exonuclease, leading to increased toxicity toward mitochondrial DNA.

A Stopped Flow Transient Kinetic Analysis of Substrate Binding and Catalysis in Escherichia coli d-3-Phosphoglycerate Dehydrogenase

Pre-steady state, stopped flow analysis of Escherichia coli d-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, α-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD+ reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD+ reduction (3.8 s–1) is similar to the kcat of the enzyme (2–3 s–1) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a VH/VD = 1.07 ± 0.06. None of the other rates determined by stopped flow analysis could account for the kcat of the enzyme in either direction (forward kcat = 0.01 s–1, reverse kcat = 2–3 s–1), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.

Comparison of thiaminase activity in fish using the radiometric and 4-nitrothiophenol colorimetric methods

ABSTRACT Thiaminase induced thiamine deficiency occurs in fish, humans, livestock and wild animals. A non-radioactive thiaminase assay was described in 2007, but a direct comparison with the radioactive 14C-thiamine method which has been in use for more than 30 years has not been reported. The objective was to measure thiaminase activity in forage fish (alewife Alosa pseudoharengus, rainbow smelt Osmerus mordax, and slimy sculpin Cottus cognatus) consumed by predators that manifest thiamine deficiency using both methods. Modifications were made to the colorimetric assay to improve repeatability. Modification included a change in assay pH, enhanced sample clean-up, constant assay temperature (37 °C), increase in the concentration of 4-nitrothiophenol (4NTP) and use of a spectrophotometer fitted with a 0.2 cm cell. A strong relationship between the two assays was found for 51 alewife (R2 = 0.85), 36 smelt (R2 = 0.87) and 20 sculpin (R2 = 0.82). Thiaminase activity in the colorimetric assay was about 1000 times higher than activity measured by the radioactive method. Application of the assay to fish species from which no thiaminase activity has previously been reported resulted in no 4NTP thiaminase activity being found in bloater Coregonus hoyi, lake trout Salvelinus namaycusch, steelhead trout Oncorhynchus mykiss or Chinook salmon Oncorhynchus tshawytscha. In species previously reported to contain thiaminase, 4NTP thiaminase activity was measured in bacteria Paenibacillus thiaminolyticus, gizzard shad Dorosoma cepedianum, bracken fern Pteridium aquilinum, quagga mussel Dreissena bugensis and zebra mussels D. polymorpha.