The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

Allylation and Alkylation of Biologically Relevant Nucleophiles by Diallyl Sulfides.

Allyl sulfides are bioactive phytochemicals found in garlic, onion, and other members of the genus Allium. Here we showed that diallyl disulfide and diallyl trisulfide can transfer allyl side chains to low molecular weight thiols. Diallyl monosulfide is inert with respect to this allyl transfer reaction. On the other hand, diallyl sulfone, a known metabolite of diallyl monosulfide, alkylates both amines and thiols under physiologically relevant conditions via isomerization to an electrophilic vinyl sulfone.

A Catalytic Role for C-H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD.

DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually take place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the C-H/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair.

Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes.

Reactions of 1,3-Diketones with a Dipeptide Isothiazolidin-3-one: Toward Agents That Covalently Capture Oxidized Protein Tyrosine Phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of type 2 diabetes; however, the enzyme has been classified by some as an "undruggable target". Here we describe studies directed toward the development of agents that covalently capture the sulfenyl amide "oxoform" of PTP1B generated during insulin signaling events. The sulfenyl amide residue found in oxidized PTP1B presents a unique electrophilic sulfur center that may be exploited in drug and probe design. Covalent capture of oxidized PTP1B could permanently disable the intracellular pool of enzyme involved in regulation of insulin signaling. Here, we employed a dipeptide model of oxidized PTP1B to investigate the nucleophilic capture of the sulfenyl amide residue by structurally diverse 1,3-diketones. All of the 1,3-diketones examined here reacted readily with the electrophilic sulfur center in the sulfenyl amide residue to generate stable covalent attachments. Several different types of products were observed, depending upon the substituents present on the 1,3-diketone. The results provide a chemical foundation for the development of agents that covalently capture the oxidized form of PTP1B generated in cells during insulin signaling events.

Toward hypoxia-selective DNA-alkylating agents built by grafting nitrogen mustards onto the bioreductively activated, hypoxia-selective DNA-oxidizing agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine).

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a heterocyclic di-N-oxide that undergoes enzymatic deoxygenation selectively in the oxygen-poor (hypoxic) cells found in solid tumors to generate a mono-N-oxide metabolite. This work explored the idea that the electronic changes resulting from the metabolic deoxygenation of tirapazamine analogues might be exploited to activate a DNA-alkylating species selectively in hypoxic tissue. Toward this end, tirapazamine analogues bearing nitrogen mustard units were prepared. In the case of the tirapazamine analogue 18a bearing a nitrogen mustard unit at the 6-position, it was found that removal of the 4-oxide from the parent di-N-oxide to generate the mono-N-oxide analogue 17a did indeed cause a substantial increase in reactivity of the mustard unit, as measured by hydrolysis rates and DNA-alkylation yields. Hammett sigma values were measured to quantitatively assess the magnitude of the electronic changes induced by metabolic deoxygenation of the 3-amino-1,2,4-benzotriazine 1,4-dioxide heterocycle. The results provide evidence that the 1,2,4-benzotiazine 1,4-dioxide unit can serve as an oxygen-sensing prodrug platform for the selective unmasking of bioactive agents in hypoxic cells.

Crystal structure of N-(quinolin-6-yl)hydroxyl-amine.

The title compound, C9H8N2O, crystallized with four independent mol-ecules in the asymmetric unit. The four mol-ecules are linked via one O-H⋯N and two N-H⋯N hydrogen bonds, forming a tetra-mer-like unit. In the crystal, mol-ecules are further linked by O-H⋯N and N-H⋯O hydrogen bonds forming layers parallel to (001). These layers are linked via C-H⋯O hydrogen bonds and a number of weak C-H⋯π inter-actions, forming a three-dimensional structure. The crystal was refined as a non-merohedral twin with a minor twin component of 0.319.

Thiol-dependent recovery of catalytic activity from oxidized protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low-molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively inactivated PTPs (PTP1B and SHP-2) by various low-molecular weight thiols and the enzyme thioredoxin. All monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. In general, molecules bearing low-pKa thiol groups were particularly effective. The biological thiol glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M(-1) s(-1), while the dithiol dithiothreitol (DTT) displayed an apparent second-order rate constant of 0.325 ± 0.007 M(-1) s(-1). The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 µM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10(-3) s(-1). The rates at which these agents regenerated oxidized PTP1B followed the order Trx > DTT > GSHand comparable values observed at 2 µM Trx, 4 mM DTT, and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M(-1) s(-1). We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low-molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.

The biological buffer bicarbonate/CO2 potentiates H2O2-mediated inactivation of protein tyrosine phosphatases.

Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H(2)O(2)-mediated cellular signal transduction processes, but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer bicarbonate/CO(2) potentiates the ability of H(2)O(2) to inactivate PTPs. The results of biochemical experiments and high-resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H(2)O(2) with HCO(3)(-)/CO(2).

Redox regulation of protein tyrosine phosphatases: methods for kinetic analysis of covalent enzyme inactivation.

Phosphorylation of tyrosine residues is an important posttranslational modification that modulates the function of proteins involved in many important cell signaling pathways. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) work in tandem to control the phosphorylation status of target proteins. Not surprisingly, the activity of some PTPs is regulated as part of the endogenous cellular mechanisms for controlling the intensity and duration of responses to various stimuli. One important mechanism for the regulation of PTPs involves endogenous production of hydrogen peroxide (H2O2) that inactivates enzymes via covalent modification of an active site cysteine thiolate group. Other endogenous metabolites and xenobiotics that inactivate PTPs via covalent mechanisms also have the potential to modulate signal transduction pathways and may possess either therapeutic or toxic properties. This chapter discusses methods for quantitative kinetic analysis of covalent inactivation of PTPs by small molecules.

Redox regulation of protein tyrosine phosphatases: structural and chemical aspects.

Protein tyrosine phosphatases (PTPs) are important targets of the H(2)O(2) that is produced during mammalian signal transduction. H(2)O(2)-mediated inactivation of PTPs also may be important in various pathophysiological conditions involving oxidative stress. Here we review the chemical and structural biology of redox-regulated PTPs. Reactions of H(2)O(2) with PTPs convert the catalytic cysteine thiol to a sulfenic acid. In PTPs, the initially generated sulfenic acid residues have the potential to undergo secondary reactions with a neighboring amide nitrogen or cysteine thiol residue to yield a sulfenyl amide or disulfide, respectively. The chemical mechanisms by which formation of sulfenyl amide and disulfide linkages can protect the catalytic cysteine residue against irreversible overoxidation to sulfinic and sulfonic oxidation states are described. Due to the propensity for back-door and distal cysteine residues to engage with the active-site cysteine after oxidative inactivation, differences in the structures of the oxidatively inactivated PTPs may stem, to a large degree, from differences in the number and location of cysteine residues surrounding the active site of the enzymes. PTPs with key cysteine residues in structurally similar locations may be expected to share similar mechanisms of oxidative inactivation.