Analytical validation of a novel panel of biomarkers for a test for preeclampsia.

Preeclampsia is a serious condition responsible for much pregnancy-related morbidity and mortality. Diagnosis of preeclampsia is difficult due to the non-specific and subjective nature of symptoms of the disease. To reduce the subjective decision making and management of preeclampsia, we identified a panel of biomarkers representing multiple and different pathogenic pathways implicated in the etiology of preeclampsia, and developed a test referred to as Preecludia™.

Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue.

DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase.

Transient Kinetic Methods for Mechanistic Characterization of DNA Binding and Nucleotide Flipping.

Enzymes that modify nucleobases in double-stranded genomic DNA, either as part of a DNA repair pathway or as an epigenetic modifying pathway, adopt a multistep pathway to locate target sites and reconfigure the DNA to gain access. Work on several different enzymes has shown that in almost all cases base flipping, also known as nucleotide flipping, is a key feature of specific site recognition. In this chapter, we discuss some of the strategies that can be used to perform a kinetic characterization for DNA binding and nucleotide flipping.

Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.

Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage.

Structure-based efforts to optimize a non-β-lactam inhibitor of AmpC β-lactamase.

β-Lactams are the most widely prescribed class of antibiotics, yet their efficacy is threatened by expression of β-lactamase enzymes, which hydrolyze the defining lactam ring of these antibiotics. To overcome resistance due to β-lactamases, inhibitors that do not resemble β-lactams are needed. A novel, non-β-lactam inhibitor for the class C β-lactamase AmpC (3-[(4-chloroanilino)sulfonyl]thiophene-2-carboxylic acid; Ki 26μM) was previously identified.

Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe.

Homoisocitrate dehydrogenase (HICDH) catalyzes the conversion of homoisocitrate to 2-oxoadipate, the third enzymatic step in the α-aminoadipate pathway by which lysine is synthesized in fungi and certain archaebacteria. This enzyme represents a potential target for anti-fungal drug design. Here, we describe the first crystal structures of a fungal HICDH, including structures of an apoenzyme and a binary complex with a glycine tri-peptide.

Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps.