The Arabidopsis dwarf1 Mutant Is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis1

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22α-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism.

Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro.

The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Mechanistic Importance of Redox Potentials and Conformational Flexibility in Electron-Transferring Flavoproteins

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated. ETF-QO contains one [4Fe-4S]2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. FAD redox potentials were measured by potentiometric titration probed by electron paramagnetic resonance (EPR) spectroscopy. The N338T and N338A mutations lowered the midpoint potentials, which resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore it is proposed that the iron-sulfur cluster is the immediate acceptor from ETF. It has been proposed that the αII domain of ETF is mobile, allowing promiscuous interactions with structurally different partners. Double electron-electron resonance (DEER) was used to measure the distance between spin labels at various sites and an enzymatically reduced FAD cofactor in Paracoccus denitrificans ETF. Two or three interspin distance distributions were observed for spin-labels in the αI (A43C) and βIII (A111C) domains, but only one is observed for a label in the βII (A210C) domain. This suggests that the αII domain adopts several stable conformations which may correspond to a closed/inactive conformation and an open/active conformation. An additional mutation, E162A, was introduced to increase the mobility of the αII domain. The E162A mutation doubled the activity compared to wild-type and caused the distance distributions to become wider. The DEER method has the potential to characterize conformational changes in ETF that occur when it interacts with various redox partners.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at -2 and -278 mV (with a ratio of 1e(-):3e(-)) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were -20 and -254 mV. All redox responses exhibit a pH dependence of approximately -59 mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry. (c) 2006 Elsevier B.V. All rights reserved.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Nicotinamide Benzimidazolide Dinucleotides, Non-Cyclisable Analogues of NAD+

Benzimidazole-based nucleotides and dinucleotides have been synthesised to increase the range of chemical tools available to probe the NAD+ biology space. They were examined for their reactivity in alkylation-type reactions, where they yielded unstable alkylated heteoaromatic adducts, both chemically and enzymatically. While unsuited for NAD+ cyclases, these NAD+ analogues could be viable substrates for non-adenine modifying NAD+-dependent enzyme classes.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Zero- one- and two-dimensional hydrogen-bonded structures in the 1:1 proton-transfer compounds of 4,5-dichlorophthalic acid with the monocyclic heteroaromatic Lewis bases 2-aminopyrimidine, nicotinamide and isonicotinamide

The structures of the anhydrous 1:1 proton-transfer compounds of 4,5-dichlorophthalic acid (DCPA) with the monocyclic heteroaromatic Lewis bases 2-aminopyrimidine, 3-(aminocarboxy) pyridine (nicotinamide) and 4-(aminocarbonyl) pyridine (isonicotinamide), namely 2-aminopyrimidinium 2-carboxy-4,5-dichlorobenzoate C4H6N3+ C8H3Cl2O4- (I), 3-(aminocarbonyl) pyridinium 2-carboxy-4,5-dichlorobenzoate C6H7N2O+ C8H3Cl2O4- (II) and the unusual salt adduct 4-(aminocarbonyl) pyridinium 2-carboxy-4,5-dichlorobenzoate 2-carboxymethyl-4,5-dichlorobenzoic acid (1/1/1) C6H7N2O+ C8H3Cl2O4-.C9H6Cl2O4 (III) have been determined at 130 K. Compound (I) forms discrete centrosymmetric hydrogen-bonded cyclic bis(cation--anion) units having both R2/2(8) and R2/1(4) N-H...O interactions. In compound (II) the primary N-H...O linked cation--anion units are extended into a two-dimensional sheet structure via amide-carboxyl and amide-carbonyl N-H...O interactions. The structure of (III) reveals the presence of an unusual and unexpected self-synthesized methyl monoester of the acid as an adduct molecule giving one-dimensional hydrogen-bonded chains. In all three structures the hydrogen phthalate anions are

The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Hepatitis C virus (HCV ) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site(IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly.

Structural Polymorphism of the Nucleosomal DNA and Implications for Protein Binding

A nucleosome forms a basic unit of the chromosome structure. A biologically relevant question is how much of the nucleosomal conformational space is accessible to protein-free DNA, and what proportion of the nucleosomal conformations are induced by bound histones. To investigate this, we have analysed high resolution xray crystal structure datasets of DNA in protein-free as well as protein-bound forms, and compared the dinucleotide step parameters for the two datasets with those for high resolution nucleosome structures. Our analysis shows that most of the dinucleotide step parameter values for the nucleosome structures lie within the range accessible to protein-free DNA, indirectly indicating that the histone core plays more of a stabilizing role. The nucleosome structures are observed to assume smooth and nearly planar curvature, implying that ‘normal’ B-DNA like parameters can give rise to a curved geometry at the gross structural level. Different nucleosome

Studies on 1-methyl adenine transfer RNA methyltransferase of Mycobacterium smegmatis

The presence of 1-methyl adenine in transfer RNA is a feature that Mycobacterium smegmatis shares with only a few other prokaryotes. The enzyme 1-methyl adenine tRNA methyl transferase from this source has been purified and the preliminary results show the presence of two activity peaks with different substrate specificity.