Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility-mass spectrometry.

CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility-mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

The intervention effect of licorice in d-galactose induced aging rats by regulating the taurine metabolic pathway.

Licorice, an edible and officinal plant material, has attracted considerable attention for its wide range of pharmacological activities. Our previous study showed that licorice can ameliorate cognitive damage and improve oxidative stress and apoptosis in aging rats induced by d-galactose (d-gal). In this study, in order to further explore the changes of the metabolic profile during the aging process and the antiaging mechanism of licorice, the 1H NMR-based metabolomics approach was used to analyze serum and urine samples and identify a potential biomarker in d-gal induced aging rats. The results revealed that the taurine metabolic pathway was significantly correlated with the ageing process in d-gal induced rats. Furthermore, the taurine contents were significantly decreased in both the serum and urine samples of aging rats compared with the controls. At the same time, the levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD) and glutamate decarboxylase type I (GAD1), which are the key enzymes affecting the synthesis reactions, were decreased in aging rats compared with the controls. After licorice administration, the levels of taurine, CDO1 and CSAD were all significantly increased. These findings firstly demonstrated that the regulation of the taurine metabolic pathway is involved in the anti-aging effect of licorice in d-gal induced aging rats.

Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum.

l-lysine is an essential amino acid that is widely used as a food supplement for humans and animals. meso-Diaminopimelic acid decarboxylase (DAPDC) catalyzes the final step in the de novol-lysine biosynthetic pathway by converting meso-diaminopimelic acid (meso-DAP) into l-lysine by decarboxylation reaction. To elucidate its molecular mechanisms, we determined the crystal structure of DAPDC from Corynebacterium glutamicum (CgDAPDC). The PLP cofactor is bound at the center of the barrel domain and forms a Schiff base with the catalytic Lys75 residue. We also determined the CgDAPDC structure in complex with both pyridoxal 5'-phosphate (PLP) and the l-lysine product and revealed that the protein has an optimal substrate binding pocket to accommodate meso-DAP as a substrate. Structural comparison of CgDAPDC with other amino acid decarboxylases with different substrate specificities revealed that the position of the α15 helix in CgDAPDC and the residues located on the helix are crucial for determining the substrate specificities of the amino acid decarboxylases.

Mechanism and Structure of γ-Resorcylate Decarboxylase.

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn2+ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution.

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ).

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H2O2-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr•. This residue then abstracts a net hydrogen atom (H•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e-/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.

Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from Selenomonas ruminantium.

Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family.

Transcriptome-guided gene isolation and functional characterization of UDP-xylose synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum caudatum Ait.

KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.

A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter.

Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of (45)Ca(2+), heat stability and Ca(2+) dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca(2+) binding transcriptional regulators.

Mechanistic studies for tri-targeted inhibition of enzymes involved in cholesterol biosynthesis by green tea polyphenols.

In the present study, we found that three enzymes, MVK, MDD and FPPS, in the mevalonate pathway (MVP) of cholesterol biosynthesis, can be simultaneously inhibited by two green tea polyphenols ((-)-epicatechin-3-gallate, ECG; (-)-epigallocatechin-3-gallate, EGCG). Molecular dynamics simulations and pharmacophore studies were carried out to elucidate the tri-targeted inhibition mechanisms. Our results indicate that similar triangular binding pockets exist in all three enzymes, which is essential for their binding with polyphenols. Two distinct binding poses for ECG and EGCG were observed in our MD simulations. These results shed light on the potential for further selective and multi-targeted inhibitor design for the treatment of hyperlipidemia.

Real-time NMR monitoring of intermediates and labile products of the bifunctional enzyme UDP-apiose/UDP-xylose synthase.

The conversion of UDP-alpha-d-glucuronic acid to UDP-alpha-d-xylose and UDP-alpha-d-apiose by a bifunctional potato enzyme UDP-apiose/UDP-xylose synthase was studied using real-time nuclear magnetic resonance (NMR) spectroscopy. UDP-alpha-d-glucuronic acid is converted via the intermediate uridine 5'-beta-l-threo-pentapyranosyl-4''-ulose diphosphate to UDP-alpha-d-apiose and simultaneously to UDP-alpha-d-xylose. The UDP-alpha-d-apiose that is formed is unstable and is converted to alpha-d-apio-furanosyl-1,2-cyclic phosphate and UMP. High-resolution real-time NMR spectroscopy is a powerful tool for the direct and quantitative characterization of previously undetected transient and labile components formed during a complex enzyme-catalyzed reaction.

Mutation and inhibition studies of mevalonate 5-diphosphate decarboxylase.

Mevalonate 5-diphosphate decarboxylase plays an important role in regulating cholesterol biosynthesis, which was studied through incubation with various synthetic substrate analogs and characterization of mutated enzymes. The results are potentially useful for further developing inhibitors that block the mevalonate pathway which is a drug target for treating cardiovascular disease and cancer.

Expression, crystallization and preliminary X-ray crystallographic analysis of human agmatinase.

Agmatine, which results from the decarboxylation of L-arginine by arginine decarboxylase, is a metabolic intermediate in the biosynthesis of putresine and higher polyamines (spermidine and spermine). Recent studies indicate that agmatine can have several important biochemical effects in humans, ranging from effects on the central nervous system to cell proliferation in cancer and viral replication. Agmatinase catalyses the hydrolysis of agmatine to putresine and urea and is a major target for drug action and development. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Human agmatinase (residues Ala36-Val352) has been overexpressed as a fusion with both N- and C-terminal purification tags in Escherichia coli and crystallized in the presence of Mn2+ and 1,6-diaminohexane at 297 K using polyethylene glycol 4000 as a precipitant. X-ray diffraction data were collected at 100 K to 2.49 A from a flash-frozen crystal. The crystals are tetragonal, belonging to space group P4(2), with unit-cell parameters a = b = 114.54, c = 125.65 A, alpha = beta = gamma = 90 degrees. Three monomers are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.66 A3 Da(-1) and a solvent content of 66.4%.

Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum.

In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum-infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 +/- 19 microM and a VMAX of 680 +/- 49 nmol of phosphatidylethanolamine formed h-1 mg-1 protein. Site-directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits (alpha and beta) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.

Identification and expression of a cDNA encoding human alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD). A key enzyme for the tryptophan-niacine pathway and "quinolinate hypothesis".

Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called "quinolinate hypothesis." Quinolinate is non-enzymatically derived from alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS). Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is the only known enzyme that can process ACMS to a benign catabolite and thus prevent the accumulation of quinolinate from ACMS. ACMSD seems to be regulated by nutritional and hormonal signals, but its molecular mechanism has, to date, been largely unknown. Utilizing partial amino acid sequences obtained from highly purified porcine kidney ACMSD, a cDNA encoding human ACMSD was cloned and characterized. The cDNA encodes a unique open reading frame of 336 amino acids and displays little homology to any known enzymes or motifs in mammalian databases, suggesting that ACMSD may contain a new kind of protein fold. Real-time PCR-based quantification of ACMSD revealed very low but significant levels of the expression in the brain. Brain ACMSD messages were down- and up-regulated in response to low protein diet and streptozocin-induced diabetes, respectively. The enzyme activities measured from partially purified brains were closely correlated with the changes in the message levels. Expression of quinolinate phosphoribosyltransferase (QPRT), another enzyme that catabolizes quinolinate, was also found in the brain. This suggests that a pathway does exist by which the levels of quinolinate in the brain are regulated. In this report, we address the molecular basis underlying quinolinate metabolism and the regulation of ACMSD expression.

Cloning and characterization of the 5'-flanking region of the oxalate decarboxylase gene from Flammulina velutipes.

The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina velutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548-23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5'-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the -580 bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5'GCGGGGTCGCCGA3', termed low pH responsive element (LPRE), corresponding to the -287 to -275 bp region of the OXDC promoter. Our results suggest that in F. velutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.

Cloning of a novel retinoid-inducible serine carboxypeptidase from vascular smooth muscle cells.

Retinoids block smooth muscle cell (SMC) proliferation and attenuate neointimal formation after vascular injury, presumably through retinoid receptor-mediated changes in gene expression. To identify target genes in SMC whose encoded proteins could contribute to such favorable biological effects, we performed a subtractive screen for retinoid-inducible genes in cultured SMC. Here, we report on the cloning and initial characterization of a novel retinoid-inducible serine carboxypeptidase (RISC). Expression of RISC is low in cultured SMC but progressively increases over a 5-day time-course treatment with all-trans-retinoic acid. A near full-length rat RISC cDNA was cloned and found to have a 452-amino acid open reading frame containing an amino-terminal signal sequence, followed by several conserved domains comprising the catalytic triad common to members of the serine carboxypeptidase family. In vitro transcription and translation experiments showed that the rat RISC cDNA generates an approximately 51-kDa protein. Confocal immunofluorescence microscopy of COS-7 cells transiently transfected with a RISC-His tag plasmid revealed cytosolic localization of the fusion protein. Western blotting studies using conditioned medium from transfected COS-7 cells suggest that RISC is a secreted protein. Tissue Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues analyzed; high level RISC expression was also observed in human kidney. In situ hybridization verified the localization of RISC to medial SMC of the adult rat aorta. Interestingly, expression in kidney was restricted to proximal convoluted tubules; little or no expression was observed in glomerular cells, distal convoluted and collecting tubules, or medullary cells. Radiation hybrid mapping studies placed the rat RISC locus on chromosome 10q. These studies reveal a novel retinoid-inducible protease whose activity may be involved in vascular wall and kidney homeostasis.

Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme.

The established pathways from serine to ethanolamine are indirect and involve decarboxylation of phosphatidylserine. Here we show that plants can decarboxylate serine directly. Using a radioassay based on ethanolamine (Etn) formation, pyridoxal 5'-phosphate-dependent l-serine decarboxylase (SDC) activity was readily detected in soluble extracts from leaves of diverse species, including spinach, Arabidopsis, and rapeseed. A putative Arabidopsis SDC cDNA was identified by searching GenBank for sequences homologous to other amino acid decarboxylases and shown by expression in Escherichia coli to encode a soluble protein with SDC activity. This cDNA was further authenticated by complementing the Etn requirement of a yeast psd1 psd2 mutant. In a parallel approach, a cDNA was isolated from a rapeseed library by its ability to complement the Etn requirement of a yeast cho1 mutant and shown by expression in E. coli to specify SDC. The deduced Arabidopsis and rapeseed SDC polypeptides are 90% identical, lack obvious targeting signals, and belong to amino acid decarboxylase group II. Recombinant Arabidopsis SDC was shown to exist as a tetramer and to contain pyridoxal 5'-phosphate. It does not attack d-serine, l-phosphoserine, other l-amino acids, or phosphatidylserine and is not inhibited by Etn, choline, or their phosphoesters. As a soluble, pyridoxal 5'-phosphate enzyme, SDC contrasts sharply with phosphatidylserine decarboxylases, which are membrane proteins that have a pyruvoyl cofactor.

Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism.

In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae.

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.