Porphyromonas gingivalis peptidylarginine deiminase (PAD) catalyzes the deimination of peptidylarginine residues of various peptides to produce peptidylcitrulline and ammonia. P. gingivalis is associated with adult-onset periodontitis and cardiovascular disease, and its proliferation depends on secretion of PAD. We have expressed two recombinant forms of the P. gingivalis PAD in Escherichia coli, a truncated form with a 43-amino acid N-terminal deletion and the full-length form of PAD as predicted from the DNA sequence. Both forms contain a poly-His tag and Xpress epitope at the N-terminus to aid in detection and purification. The activities and stabilities of these two forms have been evaluated. PAD is cold sensitive; it aggregates within 30 min at 4 °C, and optimal storage conditions are at 25 °C in the presence of a reducing agent. PAD is not a metalloenzyme and does not need a co-factor for catalysis or stability. Multiple L-arginine analogs, various arginine-containing peptides, and free L-arginine were used to evaluate substrate specificity and determine kinetic parameters.

Diphosphomevalonate (Mev⅐pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev⅐pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanismbased inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C 3 -methyl group of Mev⅐pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev⅐pp forms by the endogenous MK and phosphomevalonate kinase. We now report the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev⅐pp analogues revealed that the allosteric site is selective for compact, electron-rich C 3 -subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C 3 and that these patterns are explained well by changes in the volume of the C 3 R-group-binding pockets of the enzymes.Streptococcus pneumoniae, the primary cause of bacterial meningitis and pneumonia, kills over 4000 people daily worldwide and disproportionately affects children and the elderly (1, 2). Antibiotic resistance is a major problem in fighting this organism, with multiple-drug resistance rates as high as 95% in some regions (3). Although vaccines targeting the 7 or 23 most prevalent strains have shown success in reducing disease incidence in developed countries (4), there is a continual need for new antibiotic strategies to combat unvaccinated strains, which are rapidly filling the biological niches left by the vaccine (5).The mevalonate (Mev) 3 pathway is essential for the survival of S. pneumoniae in lung and serum (6). The bacterium uses this pathway to convert Mev to isopentenyl diphosphate, the "building block" of the isoprenoids: a class of 25,000 unique molecules having a wide range of biological functions. The pathway consists of three GHMP family kinases: Mev kinase (MK), phosphomevalonate kinase (PMK) and diphosphomevalonate decarboxylase (DPM-DC) ( Fig. 1) (7). Mutations that knock out genes in this pathway kill the organism in vivo, suggesting that S. pneumoniae cannot obtain the necessary precursors or downstream products from the host (6). In principle, each of the three enzymes is an antibiotic target, because inhibition of any of them prevents the production of isopentenyl d...

The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation.

Peptidylarginine deiminase (PAD), which catalyzes the deimination of the guanidino group from peptidylarginine residues, belongs to a superfamily of guanidino-group modifying enzymes that have been shown to produce an S-alkylthiouronium ion intermediate during catalysis. Thiol-directed reagents iodoacetamide and iodoacetate inactivate recombinant PAD, and substrate protects the enzyme from inactivation. Activity measurements together with peptide mapping by mass spectrometry of PAD modified in the absence and presence of substrate demonstrated that cysteine-351 is modified by iodoacetamide. The pKa value of the cysteine residue, 7.7 ± 0.2 as determined by iodoacetamide modification, agrees well with a critical pK value identified in pH rate studies. The role of cysteine-351 in catalysis was tested by site-directed mutagenesis in which the cysteine was replaced with serine to eliminate the proposed nucleophilic interaction. Binding studies carried out using fluorescence spectrometry established the structural integrity of the C351S PAD. However, the C351S PAD variant was catalytically inactive, exhibiting <0.01% wild-type activity. These results indicate that Cys 351 is a nucleophile that initiates the enzymatic reaction.