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PubMed2006

The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins.

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.

Amino Acid Sequence↗

PubMed1974

Genetic basis of nutritional requirements in Lactobacillus casei.

In a study of the genetic basis of multiple nutritional requirements in Lactobacillus casei, systematic attempts were made to isolate mutants that can grow in the absence of a specific nutrient required by the parental organism. Such mutants have successfully been isolated with respect to seven of twelve amino acids (aspartic acid, leucine, isoleucine, lysine, methionine, serine, and threonine) and three of four vitamins (pantothenic acid, nicotinic acid, and pyridoxal) tested, after extensive screenings employing various mutagens. Mutants that can grow without tryptophan were not isolated, but those that can grow on anthranilate or indole as well as on tryptophan were obtained at a frequency expected for single-step mutations. Activity of tryptophan synthetase was demonstrated in extracts of these anthranilate-utilizing mutants, but not in the parental strain. These results suggest that the multiple nutritional requirements of L. casei are often, if not always, due to one or a few small lesions such as base substitution mutations rather than large deletions affecting the genes involved in each biosynthetic pathway. The data would also imply that many of the biosynthetic pathways that are not fully functional in L. casei were active at one time and became nonfunctional during evolution of the present species.

Adenine↗

PubMed1991

Functional significance of overlapping transcripts of crtEF, bchCA, and puf photosynthesis gene operons in Rhodobacter capsulatus.

The Rhodobacter capsulatus crtEF, bchCA, and puf operons each encode products that function together to produce photosynthetic pigment-protein complexes. The crtEF operon encodes two enzymes of the carotenoid biosynthetic pathway, the bchCA operon encodes two enzymes of the bacteriochlorophyll biosynthetic pathway, and the puf operon contains six genes, four of which are known to code for proteins that bind pigment cofactors and position them in the intracytoplasmic membrane. These operons are adjacent on the chromosome and are transcribed in the same direction. Although each of these operons can be expressed from its own promoter, it is possible for transcription to begin at the crtEF promoter and continue through the bchCA and puf operons. We herein present the results of experiments which demonstrate that this overlapping transcriptional arrangement is important for establishment of normal levels of transcripts of the bchCA and puf operons and that read-through transcription from the bchCA operon contributes significantly to the expression of a pufB'::lac'Z gene fusion. Growth studies of crtF and bchC interposon mutants show that this read-through transcription is necessary for efficient transition from respiratory to photosynthetic growth modes.

Blotting, Northern↗

PubMed1999

Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols.

The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy.

Acetylglucosamine↗

PubMed2001

Irreversibility in unbranched pathways: preferred positions based on regulatory considerations.

It has been observed experimentally that most unbranched biosynthetic pathways have irreversible reactions near their beginning, many times at the first step. If there were no functional reasons for this fact, then one would expect irreversible reactions to be equally distributed among all positions in such pathways. Since this is not the case, we have attempted to identify functional consequences of having an irreversible reaction early in the pathway. We systematically varied the position of the irreversible reaction in model pathways and compared the resulting systemic behavior according to several criteria for functional effectiveness, using the method of mathematically controlled comparisons. This technique minimizes extraneous differences in systemic behavior and identifies those that are fundamental. Our results show that a pathway with an irreversible reaction located at the first step, and with all other reactions reversible, is on average better than an otherwise equivalent pathway with all reactions reversible, which in turn is on average better than an otherwise equivalent pathway with an irreversible reaction located at any step other than the first. Pathways with an irreversible first reaction and low concentrations of intermediates (one of the primary criteria for functional effectiveness) exhibit the following profile when compared to fully reversible pathways: changes in the concentration of intermediates in response to changes in the level of initial substrate are equally low, the robustness of the intermediate concentrations and of the flux is similar, the margins of stability are similar, flux is more responsive to changes in demand for end product, intermediate concentrations are less responsive to changes in demand for end product, and transient times are shorter. These results provide a functional rationale for the positioning of irreversible reactions at the beginning of unbranched biosynthetic pathways.

Homeostasis↗

PubMed1995

The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae.

The uaY gene codes for a transcriptional activator mediating the induction of a number of unlinked genes involved in purine utilization in Aspergillus nidulans. Here we present the complete genomic and cDNA nucleotide sequence of this gene. The gene contains two introns. The derived polypeptide of 1060 residues contains a typical zinc binuclear cluster domain and shows a number of similarities with the PPR1 regulatory gene of Saccharomyces cerevisiae. These similarities are most striking in the putative linker and dimerization regions following the zinc cluster. Gel-shift and DNase I footprinting experiments have been carried out for three genes subject to UaY-mediated induction. The binding sequence is 5'-TCGG-6X-CCGA, which is identical to the proposed PPR1 binding sites. Nevertheless, the identity of the base immediately 3' of the 5'-TCGG sequence clearly affects the affinity of the site. The site upstream of the uapA gene has been shown to be active in vivo. Binding to this site has been analysed by a number of interference techniques. There is an interesting chemical similarity between the co-inducer of the purine utilization pathway (uric acid) and that of the genes of the pyrimidine biosynthetic pathway (dihydroorotic acid) and we show that dihydroorotic acid can act as a poor inducer of at least one activity under UaY control. These striking similarities, together with the unique pattern of regulation of pyrimidine biosynthesis in S. cerevisiae, suggest that PPR1 evolved through recruitment into the pyrimidine biosynthetic pathway of an ancestral gene related to uaY.

Amino Acid Sequence↗

PubMed1990

Regulation of the genes for heme pathway enzymes in erythroid and in non-erythroid cells.

There are eight enzymes in the heme biosynthetic pathway and three enzymes in the heme catabolic pathway. Enzymatic defects in heme biosynthesis lead to clinical conditions termed porphyrias. cDNAs for five of the eight enzymes in the heme biosynthetic pathway and two of the three enzymes in the heme catabolic pathway have been cloned and characterized in mammalian cells. At least two enzymes exist as isozymes between erythroid and non-erythroid tissues. One is delta-aminolevulinic acid synthase (ALAS), and the erythroid and hepatic isozymes are coded by two separate genes. The other is porphobilinogen deaminase (PBGD), and both the erythroid and the non-erythroid PBGD mRNA are transcribed from a single PBGD gene by alternate transcription and splicing. There is also a significant tissue-specific control of expression of the uroporphyrinogen decarboxylase gene which is expressed as a unique mRNA in all tissues.

5-Aminolevulinate Synthetase↗

PubMed1999

Differential effects of 17alpha-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat.

Effects of 17alpha-ethinylestradiol (EE) on the neutral and acidic biosynthetic pathways of bile salt (BS) synthesis were evaluated in rats with an intact enterohepatic circulation and in rats with long-term bile diversion to induce BS synthesis. For this purpose, bile salt pool composition, synthesis of individual BS in vivo, hepatic activities, and expression levels of cholesterol 7alpha-hydroxylase (CYP7A), and sterol 27-hydroxylase (CYP27), as well as of other enzymes involved in BS synthesis, were analyzed in rats treated with EE (5 mg/kg, 3 days) or its vehicle. BS pool size was decreased by 27% but total BS synthesis was not affected by EE in intact rats. Synthesis of cholate was reduced by 68% in EE-treated rats, while that of chenodeoxycholate was increased by 60%. The recently identified Delta22-isomer of beta-muricholate contributed for 5.4% and 18.3 % (P < 0.01) to the pool in control and EE-treated rats, respectively, but could not be detected in bile after exhaustion of the pool. A clear reduction of BS synthesis was found in bile-diverted rats treated with EE, yet biliary BS composition was only minimally affected. Activity of CYP7A was decreased by EE in both intact and bile-diverted rats, whereas the activity of the CYP27 was not affected. Hepatic mRNA levels of CYP7A were significantly reduced by EE in bile-diverted rats only; CYP27 mRNA levels were not affected by EE. In addition, mRNA levels of sterol 12alpha-hydroxylase and lithocholate 6beta-hydroxylase were increased by bile diversion and suppressed by EE. This study shows that 17alpha-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats is associated with selective inhibition of the neutral pathway of bile salt (BS) synthesis. Simultaneous impairment of other enzymes in the BS biosynthetic pathways may contribute to overall effects of EE on BS synthesis.

Animals↗

PubMed1977

Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.

Alanine↗

PubMed2004

Strain improvement for fermentation and biocatalysis processes by genetic engineering technology.

Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized. Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition. Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering. How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity. Where will this emerging technology lead us in the twenty-first century?

Biotechnology↗

PubMed1997

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV.

BACKGROUND: In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. RESULTS: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations. CONCLUSIONS: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.

Bacterial Proteins↗

PubMed2006

Antileishmanial effect of 3-aminooxy-1-aminopropane is due to polyamine depletion.

The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, catalyzes the conversion of ornithine to putrescine. As the polyamine biosynthetic pathway is essential for the growth and survival of Leishmania donovani, the causative agent of visceral leishmaniasis, inhibition of the pathway is an important leishmaniacidal strategy. In the present study, we examined for the first time the effects of 3-aminooxy-1-aminopropane (APA), an ODC inhibitor, on the growth of L. donovani. APA inhibited the growth of both promastigotes in vitro and amastigotes in the macrophage model, with the 50% inhibitory concentrations being 42 and 5 microM, respectively. However, concentrations of APA up to 200 microM did not affect the viability of macrophages. The effects of APA were completely abolished by the addition of putrescine or spermidine. APA induced a significant decrease in ODC activity and putrescine, spermidine, and trypanothione levels in L. donovani promastigotes. Parasites were transfected with an episomal ODC construct, and these ODC overexpressers exhibited significant resistance to APA and were concomitantly resistant to sodium antimony gluconate (Pentostam), indicating a role for ODC overexpression in antimonial drug resistance. Clinical isolates with sodium antimony gluconate resistance were also found to overexpress ODC and to have significant increases in putrescine and spermidine levels. However, no increase in trypanothione levels was observed. The ODC overexpression in these clinical isolates alleviated the antiproliferative effects of APA. Collectively, our results demonstrate that APA is a potent inhibitor of L. donovani growth and that its leishmaniacidal effect is due to inhibition of ODC.

Animals↗

PubMed1991

Phenotypic heterogeneity in the syndromes of 3-methylglutaconic aciduria.

Combined 3-methylglutaconic and 3-methylglutaric aciduria, one of the more common urinary organic acid abnormalities, has been observed in at least three clinical syndromes. We studied an additional seven patients with 3-methylglutaconic aciduria, four of whom were best categorized as having the type II syndrome, two as having an "unspecified" syndrome, and one who may have had a primary urea cycle defect. In cultured cells and autopsy tissues derived from patients with the type II and unspecified syndromes, we were unsuccessful in identifying a defect in the leucine degradative pathway distal to 3-methylcrotonyl-coenzyme A carboxylase and in the cholesterol biosynthetic pathway between 3-hydroxy-3-methylglutaryl-coenzyme A reductase and diphosphomevalonate decarboxylase. Further assessment of the cholesterol biosynthetic pathway in several patients with one of the two types of disease also provided no defined abnormality. The primary metabolic defects in the type II and unspecified syndromes remain undefined.

Amino Acid Metabolism, Inborn Errors↗

PubMed2003

Production of lipid compounds in the yeast Saccharomyces cerevisiae.

This review describes progress using the yeast Saccharomyces cerevisiae as a model organism for the fast and efficient analysis of genes and enzyme activities involved in the lipid biosynthetic pathways of several donor organisms. Furthermore, we assess the impact of baker's yeast on the production of novel, high-value lipid compounds. Yeast can be genetically modified to produce selected substances in relatively high amounts. A major advantage in choosing yeast as an object for metabolic engineering is the fact that the lipid pathways in this organism have been described in detail and are well characterized. We focus on the de novo production of three major families of lipid products. These are: (1) sterols, providing some previously known and some novel applications as examples of the lipid pathway enhancement that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega-3 and omega-6 fatty acids which do not occur naturally in yeast. We utilize the current knowledge and point out perspectives and problems for future biotechnological applications in the field of lipid compounds.

Adrenal Cortex Hormones↗

PubMed2005

Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis.

Betaine is an important osmoprotectant, synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline. Recently, a biosynthetic pathway of betaine from glycine, catalyzed by two N-methyltransferase enzymes, was found. Here, the potential role of N-methyltransferase genes for betaine synthesis was examined in a freshwater cyanobacterium, Synechococcus sp. PCC 7942, and in Arabidopsis plants. It was found that the coexpression of N-methyltransferase genes in Synechococcus caused accumulation of a significant amount of betaine and conferred salt tolerance to a freshwater cyanobacterium sufficient for it to become capable of growth in seawater. Arabidopsis plants expressing N-methyltransferase genes also accumulated betaine to a high level in roots, stems, leaves, and flowers and improved seed yield under stress conditions. Betaine levels were higher than those produced by choline-oxidizing enzymes. These results demonstrate the usefulness of glycine N-methyltransferase genes for the improvement of abiotic stress tolerance in crop plants.

Arabidopsis↗

PubMed2002

Cooperative binding of the cytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy.

Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative complex composed of Apg1 kinase and several interacting proteins that are specific for either the Cvt pathway or autophagy. A second required complex includes a phosphatidylinositol (PtdIns) 3-kinase and associated proteins that are involved in its activation and localization. The majority of proteins required for the Cvt and autophagy pathways localize to a perivacuolar pre-autophagosomal structure. We show that the Cvt13 and Cvt20 proteins are required for transport of precursor Ape1 through the Cvt pathway. Both proteins contain phox homology domains that bind PtdIns(3)P and are necessary for membrane localization to the pre-autophagosomal structure. Functional phox homology domains are required for Cvt pathway function. Cvt13 and Cvt20 interact with each other and with an autophagy-specific protein, Apg17, that interacts with Apg1 kinase. These results provide the first functional connection between the Apg1 and PtdIns 3-kinase complexes. The data suggest a role for PtdIns(3)P in the Cvt pathway and demonstrate that this lipid is required at the pre-autophagosomal structure.

Amino Acid Sequence↗

PubMedDate not supplied

Identification of a lysA-like gene required for tabtoxin biosynthesis and pathogenicity in Pseudomonas syringae pv. tabaci strain PTBR2.024.

Pseudomonas syringae pv. tabaci strain PTBR2.024 produces tabtoxin and causes wildfire disease on tobacco and green bean. PTBR7.000, a Tn5 mutant of PTBR2.024, does not produce tabtoxin, is nonpathogenic on tobacco, and is prototrophic. A 3-kb fragment from a genomic library of the parent strain PTBR2.024 complemented both mutant phenotypes. This 3-kb fragment contains two open reading frames (ORFs), ORF1 and ORF2, and two truncated ORFs, ORF3 and ORF4. The Tn5 insert in PTBR7.000 was mapped to ORF2, and complementation studies showed that an intact ORF2 was sufficient to restore tabtoxin production and pathogenicity. The deduced amino acid sequences of ORF2 and truncated ORF3 contain significant homology to bacterial lysine biosynthetic enzymes, diaminopimelate decarboxylase, and delta 1-piperidine-2,6-dicarboxylate succinyl transferase, respectively. ORF2, however, is not required for lysine biosynthesis. We designated the sequence corresponding to ORF2 as gene tabA and propose that the product of tabA is an enzyme in the tabtoxin biosynthetic pathway that recognizes a substrate analogue of a compound in the lysine biosynthetic pathway.

Acyltransferases↗

PubMed2003

Biosynthesis and metabolism of brassinosteroids.

Brassinosteroids (BRs) are steroid hormones that regulate the growth and development of plants. Detailed study of the biosynthesis of brassinolide, a C28 BR, revealed that two parallel routes, the early and late C-6 oxidation pathways, are connected at multiple steps and also are linked to the early C-22 oxidation pathway. Thus, BR biosynthetic pathways are highly networked. Furthermore, the biosynthesis of C27 BRs was shown to proceed in a similar way to that of C28 BRs. Information on enzymes and genes involved in the BR biosynthesis, as well as their regulation, has been obtained using BR-deficient and BR-insensitive mutants. In addition, the biosynthesis of sterols, which were recently recognized not only as precursors of BRs and membrane constituents, but also as modulators of plant development, is discussed. Various metabolic reactions of BRs including epimerization, oxidation, and conjugation are also summarized.

Brassinosteroids↗