Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase (TS) to catalyze the methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP). All known thymidylate synthases (TSs) rely on an active site residue of the enzyme to activate dUMP1, 2. This functionality has been demonstrated for classical TSs, including human TS, and is instrumental in mechanism-based inhibition of these enzymes. Here we report the first example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3–5. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (i.e. a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2’-deoxythymidine-5’-monophosphate (dTMP). These observations indicate a very different chemical cascade than that of classical TSs or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Since several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg 2؉ ions for catalysis and that an essential Mg 2؉ ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg 2؉ changes along the reaction coordinate. At saturating concentrations of ATP and Mg 2؉ ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg 2؉ , the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.Breaks in DNA result from spontaneous hydrolysis of the phosphodiester backbone and are formed as transient intermediates during DNA replication and repair pathways. Mammals have three genes encoding DNA ligases that seal these breaks and restore the continuous nature of chromosomes. DNA ligase I (LIG1) 2 is essential for ligation of single-strand breaks in the nucleus, including the ligation of Okazaki fragments during discontinuous DNA replication (1). DNA ligase III (LIG3) is required for mitochondrial DNA replication and repair. Although LIG3 has been assumed to play essential roles in nuclear DNA repair, it was recently shown to be dispensable for nuclear genomic maintenance (2, 3). DNA ligase IV (LIG4) is specialized for repair of nuclear double-strand breaks and is required for nonhomologous end joining and V(D)J recombination (4, 5).The overall reaction catalyzed by DNA ligases involves the formation of a phosphodiester bond between an adjacent 3Ј-hydroxyl and a 5Ј-phosphate in DNA. The reaction proceeds via a universally conserved pathway ( Fig. 1) (6 -8). First, the apoenzyme catalyzes transfer of the AMP group from a nucleotide cofactor to an active site lysine, forming an adenylylated enzyme intermediate. Eukaryotic ligases use ATP as the adenylyl group donor, whereas bacterial ligases utilize either ATP or NAD ϩ . After binding a nicked DNA substrate, the adenylylated enzyme catalyzes transfer of the adenylyl group to the 5Ј-phosphate present at the nick, forming an adenylylated DNA intermediate. In the fina...

Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. Recently it has been shown that FDTS catalysis does not rely on an enzymatic nucleophile and the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist the efforts to rationally design inhibitors as leads for future antibiotics.

Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis.

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.

With no attempt made to influence their diet, six sedentary obese men ages 19 to 31 completed 16 weeks of vigorous walking 90 min, 5 days/week, on a treadmill at up to 3.2 mph on a 10% grade, expending about 1100 kcal per session. Body composition studies indicated a loss of 5.9 kg of body fat and a gain of 0.2 kg of lean tissue for a net loss of 5.7 kg. Percentage body fat decreased from 23.3 to 17.4. Monitored food intake initially increased, then progressively decreased below pretraining levels. Work capacity and cardiovascular efficiency improved with training. Plasma cholesterol and triglyceride concentrations were not significantly changed; however, high density lipoprotein cholesterol progressively increased to 15.6% above pretraining levels and the high/low density lipoprotein ratio increased 25.9%. Fasting blood sugar was significantly lower after training. Blood glucose concentrations after a glucose challenge did not significantly change, but a 43% reduction in plasma radioimmunoassay insulin levels and a 36% decrease in the ratio of insulin/glucose concentration occurred. Thus, vigorous regular walking resulted in a reduction of body fate stores, endogenous insulin requirements, and food intake, and perhaps improved the ability to eliminate cholestrol by increasing the plasma high density lipoprotein fraction.

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV–visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2+ moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of p-hydroxyphenylglycine, a recurrent substructure of polycyclic nonribosomal peptide antibiotics such as vancomycin. HMS has the same structural fold as and uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD) (4-hydroxyphenylpyruvate (HPP) and O(2)). Moreover, HMS catalyzes a very similar dioxygenation reaction to that of HPPD, adding the second oxygen atom to the benzylic position, rather than the aromatic C1 carbon of the substrate. The dissociation constant for HPP (59 microM) was measured under anaerobic conditions by titrating substrate with enzyme and monitoring the intensity of the weak (epsilon(475nm ) approximately 250 M(-1) cm(-1)) charge-transfer absorption band of the HMS.Fe(II).HPP complex. Pre-steady-state analysis indicates that evidence exists for the accumulation of three intermediates in a single turnover and the decay of the third is rate-limiting in multiple turnovers. The rate constants used to fit the data were k(1) = 1 x 10(5) M(-1) s(-1), k(2) = 250 s(-1), k(3) = 5 s(-1), and k(4) = 0.3 s(-1). However, the values for k(1) and k(2) could not be accurately measured due to both a prolonged mixing time for the HMS system that obscures observation at the early times (<10 ms) and the apparent high relative value of k(2). The third phase, k(3), is attributed to the formation of the product complex, and no kinetic isotope effect was observed on this step when the protons of the substrate's benzylic carbon were substituted with deuteriums, suggesting that hydroxylation is fast relative to the steps observed. The final and predominantly rate-limiting step shows a 3-fold decrease in the magnitude of the rate constant in deuterium oxide solvent, and a proton inventory for this step suggests the contribution of a single proton from the solvent environment.