The effects of partial thiamin deficiency and oxidative stress (i.e., glyoxal and methylglyoxal) on the levels of alpha-oxoaldehyde plasma protein adducts in Fischer 344 rats

We hypothesized that in marginal thiamin deficiency intracellular alpha-oxoaldehydes form macromolecular adducts that could possibly be genotoxic in colon cells; and that in the presence of oxidative stress these effects are augmented because of decreased detoxification of these aldehydes. We have demonstrated that reduced dietary thiamin in F344 rats decreased transketolase activity and increased alpha-oxoaldehyde adduct levels. The methylglyoxal protein adduct level was not affected by oral glyoxal or methylglyoxal in the animals receiving thiamin at the control levels but was markedly increased in the animals on a thiamin-reduced diet. These observations are consistent with our suggestion that the induction of aberrant crypt foci with marginally thiamin-deficient diets may be a consequence of the formation of methylglyoxal adducts.     

The role of firefly luciferase C-terminal domain in efficient coupling of adenylation and oxidative steps

The N-terminal domain (N-domain) of the firefly luciferase from Photinus pyraris has weak luminescence activity, and shows a unique light emitting profile with very long rise time of more than several minutes. Through a sensitive assay of the reaction intermediate luciferyl-adenylate (LH2-AMP), we found that the slow increase in the N-domain luminescence faithfully reflected the concentration of dissociated LH2-AMP. No such correlation was observed for wild-type or mutant enzymes with short rise time, except one with longer rise time. The results suggest that the C-terminal domain plays an indispensable role in efficiently coupling adenylation and oxidative steps.     

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.     

Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells

A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl-peptide and geranylgeranyl-peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP ( approximately 0.01 pmol). A linear response has been established over a range of 5-1000 pg ( approximately 0.01-2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125+/-0.010 pmol/10(6)cells) and GGPP (0.145+/-0.008 pmol/10(6)cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue.     

Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification

The catabolism of ATP and other nucleotides participates partly in the important function of nucleotide salvage by activated cells and also in removal or de novo generation of compounds including ATP, ADP, and adenosine that stimulate purinergic signaling. Seven nucleotide pyrophosphatase/phosphodiesterase NPP family members have been identified to date. These isoenzymes, related by up conservation of catalytic domains and certain other modular domains, exert generally non-redundant functions via distinctions in substrates and/or cellular localization. But they share the capacity to hydrolyze phosphodiester or pyrophosphate bonds, though generally acting on distinct substrates that include nucleoside triphosphates, lysophospholipids and choline phosphate esters. PP_i generation from nucleoside triphosphates, catalyzed by NPP1 in tissues including cartilage, bone, and artery media smooth muscle cells, supports normal tissue extracellular PP_i levels. Balance in PP_i generation relative to PP_i degradation by pyrophosphatases holds extracellular PP_i levels in check. Moreover, physiologic levels of extracellular PP_i suppress hydroxyapatite crystal growth, but concurrently providing a reservoir for generation of pro-mineralizing P_i. Extracellular PP_i levels must be supported by cells in mineralization-competent tissues to prevent pathologic calcification. This support mechanism becomes dysregulated in aging cartilage, where extracellular PP_i excess, mediated in part by upregulated NPP1 expression stimulates calcification. PP_i generated by NPP1modulates not only hydroxyapatite crystal growth but also chondrogenesis and expression of the mineralization regulator osteopontin. This review pays particular attention to the role of NPP1-catalyzed PP_i generation in the pathogenesis of certain disorders associated with pathologic calcification.     

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca²⁺-ATPase, V-H⁺-ATPase, H⁺-PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

Structure of a polyisoprenoid binding domain from Saccharophagus degradans implicated in plant cell wall breakdown

Saccharophagus degradans belongs to a recently discovered group of marine bacteria equipped with an arsenal of sugar cleaving enzymes coupled to carbohydrate-binding domains to degrade various insoluble complex polysaccharides. The modular Sde-1182 protein consists of a family 2 carbohydrate binding module linked to a X158 domain of unknown function. The 1.9 Å and 1.55 Å resolution crystal structures of the isolated X158 domain bound to the two related polyisoprenoid molecules, ubiquinone and octaprenyl pyrophosphate, unveil a β-barrel architecture reminiscent of the YceI-like superfamily that resembles the architecture of the lipocalin fold. This unprecedented association coupling oxidoreduction and carbohydrate recognition events may have implications for effective nutrient uptake in the marine environment.     

Increased nonsterol isoprenoids, dolichol and ubiquinone, in the Smith-Lemli-Opitz syndrome: effects of dietary cholesterol

Smith-Lemli-Opitz syndrome (SLOS) is an inherited autosomal recessive cholesterol deficiency disorder. Our studies have shown that in SLOS children, urinary mevalonate excretion is normal and reflects hepatic HMG-CoA reductase activity but not ultimate sterol synthesis. Hence, we hypothesized that in SLOS there may be increased diversion of mevalonate to nonsterol isoprenoid synthesis. To test our hypothesis, we measured urinary dolichol and ubiquinone, two nonsterol isoprenoids, in 16 children with SLOS and 15 controls, all fed a low-cholesterol diet. The urinary excretion of both dolichol (P < 0.002) and ubiquinone (P < 0.02) in SLOS children was 7-fold higher than in control children, whereas mevalonate excretion was comparable. In a subset of 12 SLOS children, a high-cholesterol diet decreased urinary mevalonate excretion by 61% (P < 0.001), dolichol by 70% (P < 0.001), and ubiquinone by 67% (P < 0.03). Our hypothesis that in SLOS children, normal urinary mevalonate excretion results from increased diversion of mevalonate into the production of nonsterol isoprenoids is supported. Dietary cholesterol supplementation reduced urinary mevalonate and nonsterol isoprenoid excretion but did not change the relative ratios of their excretion. Therefore, in SLOS, a secondary peripheral regulation of isoprenoid synthesis may be stimulated.