Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.

Assaying for the 3-hydroxypropionate cycle of carbon fixation.

The 3-hydroxypropionate cycle is a novel pathway for autotrophic CO2 fixation, which has been demonstrated in the thermophilic phototrophic bacterium Chloroflexus aurantiacus; a yet to be defined variant of this pathway occurs in autotrophic members of the Sulfolobales (Crenarchaeota). The 3-hydroxypropionate cycle consists of the conversion of acetyl-CoA to succinyl-CoA, via malonyl-CoA, 3-hydroxypropionate, propionyl-CoA, and methylmalonyl-CoA. Carboxylation of acetyl-CoA and propionyl-CoA by acetyl-CoA/propionyl-CoA carboxylase are the CO2 fixation reactions. Succinyl-CoA serves as a precursor of cell carbon and also as a precursor of the starting compound acetyl-CoA. In C. aurantiacus, the cycle is completed by converting succinyl-CoA to malyl-CoA and cleaving malyl-CoA to acetyl-CoA and glyoxylate. Glyoxylate is then converted in a second cyclic pathway to pyruvate, which serves as a universal cell carbon precursor. The fate of succinyl-CoA in Sulfolobales is at issue. Assays used to study the characteristic enzymes of this novel pathway in C. aurantiacus are reported.

Structural characterization of sialic acid synthase by electrospray mass spectrometry--a tetrameric enzyme composed of dimeric dimers.

Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis.

Stabilization and characterization of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae.

Histidine-tagged homocitrate synthase from Saccharomyces cerevisiae was purified to about 98% using a Ni-NTA resin and stabilized using a combination of 100 mM guanidine hydrochloride, 100 mM alpha-cyclodextrin, and 600 mM ammonium sulfate. The enzyme was assayed using dichlorophenol indophenol (DCPIP) as an oxidant to oxidize the CoASH produced in the reaction. A stoichiometry of 1:1 was obtained between DCPIP and CoASH. Kinetic parameters for the stable enzyme at pH 7.5 are: Km (AcCoA), 24 microM: Km (alpha-kg), 1.3 mM; and kcat, 37 min(-1). The enzyme, in the absence of reactants, self-associates, as suggested by size exclusion chromatography. Fluorescence and circular dichroic spectra suggested a partially exposed tryptophan residue and a mixed (alpha/beta) secondary structure for the enzyme. Fluorescence quenching studies with KI, CsCl, and acrylamide suggest that the microenvironment around the single tryptophan residue of the enzyme has some positive charge.

Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships.

Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus, are approximately 40% identical in sequence to the Escherichia coli 2-methylcitrate synthase and also possess 2-methylcitrate synthase activity. The data are discussed with respect to the structure, function and evolution of citrate synthase and 2-methylcitrate synthase.

Homocitrate synthase is located in the nucleus in the yeast Saccharomyces cerevisiae.

We have generated monoclonal antibodies against nuclear proteins from the yeast Saccharomyces cerevisiae. The monoclonal antibodies react with proteins of 47 and 49 kDa on immunoblots and with partially overlapping sets of proteins on two-dimensional nonequilibrium pH gradient electrophoresis-SDS blots. Immunofluorescence localization shows a nuclear staining pattern. Immunoscreening a yeast expression library yielded five independent full-length clones of two open reading frames from chromosome IV, corresponding to YDL182w (LYS20) and YDL131w in the Saccharomyces genome data base. These two open reading frames are predicted to encode homocitrate synthase isozymes of 47 and 49 kDa, respectively. A clone carrying YDL182w was sequenced in its entirety and directs the expression of a 47-kDa protein in Escherichia coli. A clone carrying YDL131w expresses a 49-kDa protein in E. coli. Yeast grown in minimal medium plus lysine show significant reductions in nuclear immunofluorescence staining. Cell fractionation studies localize the 47- and 49-kDa proteins to the nucleus. Nuclear fractionation studies reveal that a portion of the 47- and 49-kDa proteins can only be extracted with DNase digestion and high salt. The localization of homocitrate synthase to the nucleus is unexpected given previous reports that homocitrate synthase is present in mitochondria and the cytoplasm in S. cerevisiae.

Identification and characterization of phosphinothricin-tripeptide biosynthetic genes in Streptomyces viridochromogenes.

A 4-kb BamHI fragment of Streptomyces viridochromogenes Tü494 carrying phosphinothricin-tripeptide (PTT) biosynthetic genes has been identified by complementation of a nonproducing mutant which is defective in the tripeptide formation step. Nucleotide sequence analysis revealed one incomplete and three complete genes on the cloned fragment. The incomplete gene ('pms) codes for the C terminus of the phosphinomethylmalic acid synthase as determined by comparison with a region from the bialaphos biosynthetic cluster [Shimotohno et al., Agric. Biol. Chem. 54 (1990) 463-470] and with databases. Subcloning experiments showed that the juxtaposing phsA gene is sufficient to restore productivity of the blocked mutant. Analysis of gene disruption and gene replacement mutants confirmed that phsA specifies an enzyme involved in tripeptide formation. Similarities to peptide synthetases indicate that the condensation step follows a thio-template mechanism. A conserved region located in the C terminus of the PhsA protein showed identity to 4'-phosphopantetheine-binding sites of fatty acid and polyketide synthases. In the N terminus, a typical acyl transfer motif has been identified and this may be involved in transthiolation. A similar motif also appears in the deduced product of the third gene (dea), which probably catalyses the deacetylation of N-acetyl-PTT to PTT. The previously described PTT resistance-encoding gene (pat) was located between the phsA and the dea genes.

Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.

Determination of isocitrate lyase and malate synthase activities in a marine bivalve mollusk by a new method of assay.

1. A new method for the assay of isocitrate lyase (EC 4.1.3.1) was developed, based on the isolation of 14C-glyoxylate semicarbazone by co-crystallization with authentic carrier. The method was easily adapted to measure malate synthase (EC 4.1.3.2). 2. Interfering reactions were avoided with this method, and isocitrate dehydrogenase (ID) was easily distinguished from isocitrate lyase (IL). Assay of IL in germinating pumpkin seeds gave rates proportional to the amount of extract, with greater sensitivity and less variability than the spectrophotometric method. 3. Six species of marine bivalve mollusks were tested for IL activity, and two species produced glyoxylate: Crassostrea virginica at 0.10 mumol/hr/g tissue, and Petricola pholadiformis at 0.85 mumol/hr/g. The other four species, and four other marine invertebrates from other phyla, lacked detectable IL activity. 4. The rate of disappearance of glyoxylate in the malate synthase (MS) reaction indicated that Petricola had an activity of 0.60 mumol/hr/g: this is the first demonstration of activities of both IL and MS in a marine invertebrate.

The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase.

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.

Escherichia coli isocitrate lyase: properties and comparisons.

The glyoxylate cycle was first discovered during studies on bacteria and fungi with the ability to grow on acetate or ethanol as the sole carbon source. Isocitrate lyase, the first enzyme unique to the glyoxylate cycle, has been studied in numerous prokaryotic and eukaryotic organisms. However, information on this enzyme from Escherichia coli is limited. We have recently reported the purification and in vitro phosphorylation of this enzyme. In the present study we have examined and characterized a variety of inhibitors, the divalent cation requirement and the amino acid composition of E. coli isocitrate lyase and compared these results to those obtained with other organisms.

Assay of acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.