Comparative kinetic analysis of glycerol 3-phosphate cytidylyltransferase from Enterococcus faecalis and Listeria monocytogenes

Background: Glycerol 3-phosphate cytidylyltransferase (GCT) is an enzyme central to the synthesis of teichoic acids, components of the cell wall in gram positive bacteria. Catalysis by GCT from Enterococcus faecalis and Listeria monocytogenes has been investigated and catalytic properties compared. Material/Methods: The genes encoding GCT were cloned from genomic DNA and recombinant proteins expressed in E. coli and purified. Enzyme assays were used to determine kinetic constants k(cat) and K-m. Chemical crosslinking provided a means to assess quaternary structure of each GCT. Results: Recombinant Enterococcus faecalis GCT had an apparent k(cat) value of 1.51 s(-1) and apparent K-m values of 2.42 mM and 4.03 mM with respect to substrates cytidine 5\u27-triphosphate (CTP) and glycerol phosphate. Listeria monocytogenes GCT had an apparent k(cat) value of 4.15 s(-1) and apparent K-m values of 1.52 mM and 6.56 mM with respect to CTP and glycerol phosphate. This resulted in k(cat)/K-m values of 0.62 s(-1)mM(-1) and 0.37 s(-1)mM(-1) for E. faecalis GCT and 2.73 s(-1)mM(-1) and 0.63 s(-1)mM(-1) for L. monocytogenes GCT with respect to CTP and glycerol phosphate, respectively. Conclusions: The genome of both Enterococcus faecalis and Listeria monocytogenes contain a gene that encodes a functional GCT. The genes are 67% identical at the nucleotide level and the encoded proteins exhibit a 63% amino acid identity. The purified, recombinant enzymes each appear to be dimeric and display similar kinetic characteristics. Studying the catalytic characteristics of GCT isoforms from pathogenic bacteria provides information important for the future development of potential antibacterial agents

The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. The enzyme is found in eukaryotes and prokaryotes; and phylogenetic analysis has revealed two classes of HMG-CoA reductase, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of eubacteria and certain other archaea. Three-dimensional structures of the catalytic domain of HMG-CoA reductases from humans and from the bacterium Pseudomonas mevalonii, in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis. The reaction catalyzed by human HMG-CoA reductase is a target for anti-hypercholesterolemic drugs (statins), which are intended to lower cholesterol levels in serum. Eukaryotic forms of the enzyme are anchored to the endoplasmic reticulum, whereas the prokaryotic enzymes are soluble. Probably because of its critical role in cellular cholesterol homeostasis, mammalian HMG-CoA reductase is extensively regulated at the transcriptional, translational, and post-translational levels

Vanadium Complexes Are in vitro Inhibitors of Leishmania Secreted Acid Phosphatases

Leishmaniasis is a parasitic disease caused by the protozoa Leishmania. These organisms secrete acid phosphatases during their growth cycle as an important part of cell targeting to host macrophage cells thus allowing for a successful infection. Secreted acid phosphatases (SAP) are reported to play a significant role in the survival of Leishmania cells, thus evaluation of these enzymes is of interest. The inhibition of SAP can be the focus of a new drug therapy. We tested for SAP activity from Leishmania tarentolae following the addition of a series of vanadium complexes including decavanadate. Cell cultures at different stages in their growth curve were harvested by centrifugation and supernatant was collected. The SAP activity in the supernatant was assayed with the artificial substrate p-nitrophenylphosphate (pNPP). Incubation with orthovanadate resulted in a decrease in activity of 18% ± 1 relative to the control, in comparison to decavanadate, which resulted in a 35% ± 4 decrease in activity. Other vanadium complexes showed smaller inhibitory effects than orthovanadate. Some vanadium complexes appeared to have an effect on reducing cell clumping when compared to control cells. The SAP was partially isolated through anion exchange chromatography and results indicate that SAP isozyme forms are present in the supernatant from cells. Future work is focused on obtaining recombinant enzyme which can be more completely characterized for inhibition by vanadium complexes

Structures of lovastatin, a statin drug that competitively inhibits HMGR, and of HMG-CoA

<p><b>Copyright information:</b></p><p>Taken from "The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases"</p><p>Genome Biology 2004;5(11):248-248.</p><p>Published online 1 Nov 2004</p><p>PMCID:PMC545772.</p><p>Copyright © 2004 BioMed Central Ltd</p> It can be seen that the portion of the drug shown here at the top resembles the HMG portion of HMG-CoA

Methyl farnesoate synthesis in the lobster mandibular organ: The roles of HMG-CoA reductase and farnesoic acid O-methyltransferase

Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ-inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT-mRNA levels after 5 h. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar