cobB function is required for catabolism of propionate in Salmonella typhimurium LT2: evidence for existence of a substitute function for CobB within the 1,2-propanediol utilization (pdu) operon.

The cobB function of Salmonella typhimurium LT2 was defined in vivo as an alternative activity for the nicotinic acid mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme (CobT), which is involved in the assembly of the nucleotide loop of cobalamin in this bacterium (J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante-Semerena, J. Bacteriol. 176:3568-3575, 1994). In this paper we document that, independent of their inability to substitute for CobT function, cobB mutants are unable to use propionate as a carbon and energy source. A plasmid carrying only a wild-type copy of cobB complemented the cobalamin biosynthesis and propionate catabolism phenotypes of cobB mutants, indicating that a lack of CobB was responsible for both phenotypes. We demonstrate the existence of a function encoded by the 1,2-propanediol utilization (pdu) operon, which when induced by 1,2-propanediol compensated for the lack of CobB during propionate catabolism but failed to compensate for CobT in the assembly of the nucleotide loop of cobalamin in a cobB cobT double mutant.     

Purification and partial amino acid sequence of a glutamyl-tRNA synthetase from Rhizobium meliloti

A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step. Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54,000, as judged by SDS-gel electrophoresis. The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA synthase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively. The small amount of glutamyl-tRNA synthetase present in R. meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.     

Derepressed levels of glutamate synthase and glutamine synthetase in Escherichia coli mutants altered in glutamyl-transfer ribonucleic acid synthetase.

The levels of glutamate synthase and of glutamine synthetase are both derepressed 10-fold in strain JP1449 of Escherichia coli carrying a thermosensitive mutation in the glutamyl-transfer ribonucleic acid (tRNA) synthetase and growing exponentially but at a reduced rate at a partially restrictive temperature, compared with the levels in strain AB347 isogenic with strain JP1449 except for this thermosensitive mutation and the marker aro. These two enzymes catalyze one of the two pathways for glutamate biosynthesis in E. coli, the other being defined by the glutamate dehydrogenase. We observed a correlation between the percentage of charged tRNAGlu and the level of glutamate synthase in various mutants reported to have an altered glutamyl-tRNA synthetase activity. These results suggest that a glutamyl-tRNA might be involved in the repression of the biosynthesis of the glutamate synthase and of the glutamine synthetase and would couple the regulation of the biosynthesis of these two enzymes, which can work in tandem to synthesize glutamate when the ammonia concentration is low in E. coli but whose structural genes are quite distant from each other. No derepression of the level of the glutamate dehydrogenase was observed in mutant strain JP1449 under the conditions where the levels of the glutamine synthetase and of the glutamate synthase were derepressed. This result indicates that the two pathways for glutamate biosynthesis in E. coli are under different regulatory controls. The glutamate has been reported to be probably the key regulatory element of the biosynthesis of the glutamate dehydrogenase. Our results indicate that the cell has chosen the level of glutamyl-tRNA as a more sensitive probe to regulate the biosynthesis of the enzymes of the other pathway, which must be energized at a low ammonia concentration.     

Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis.

The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.     

Detection of putative peptide synthetase genes in Trichoderma species: application of this method to the cloning of a gene from T. harzianum CECT 2413

Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.     

Phosphopantothenoylcysteine synthetase from Escherichia coli. Identification and characterization of the last unidentified coenzyme A biosynthetic enzyme in bacteria

Phosphopantothenoylcysteine synthase catalyzes the formation of (R)-4'-phospho-N-pantothenoylcysteine from 4'-phosphopantothenate and l-cysteine: this enzyme, involved in the biosynthesis of coenzyme A (CoA), has not previously been identified. Recently it was shown that the NH(2)-terminal domain of the Dfp protein from bacteria catalyzes the next step in CoA biosynthesis, the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to form 4'-phosphopantetheine (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). We have partially purified phosphopantothenoylcysteine decarboxylase from Escherichia coli and demonstrated that the protein encoded by the dfp gene, here renamed coaBC, also has phosphopantothenoylcysteine synthetase activity, using CTP rather than ATP as the activating nucleoside 5'-triphosphate. This discovery completes the identification of all the enzymes involved in the biosynthesis of coenzyme A in bacteria.     

Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.     

Nostophycin biosynthesis is directed by a hybrid polyketide synthase-nonribosomal peptide synthetase in the toxic cyanobacterium Nostoc sp. strain 152

Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.     

Structural analysis of a peptide synthetase gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii.

Lysergyl peptide synthetase 1 catalyzes the assembly of toxic ergopeptines from activated D-lysergic acid and three amino acids. The gene encoding this enzyme in the endophytic fungus Neotyphodium lolii was analyzed and compared to a homologous gene from the ergot fungus Claviceps purpurea. Each gene contained two introns, which were found in the same relative position within two modules of the gene. The 5' ends of the two genes were unusually divergent. Signature sequences determining substrate specificity were similar in adenylation domains that recognized identical amino acids but differed within the adenylation domain for the amino acid that varies between the major ergopeptines of the two fungi. Homologues were detected in several related endophytic fungi; the tall fescue endophyte Neotyphodium coenophialum contained a divergent, second copy of the gene. Our results provide new information on the structure and distribution of this important peptide synthetase involved in ergot alkaloid biosynthesis.     

Determination of the minimal fragment of the poliovirus IRES that is necessary for the formation of a specific complex with the human glycyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charge a tRNA molecule with a cognate amino acid that is required for protein synthesis. Glycyl-tRNA synthetase is one of the most interesting aminoacyl-tRNA synthetases due to its structural variability and functional features in different organisms. Recently, it has been shown that human glycyl-tRNA synthetase is a regulator of translational initiation of poliovirus mRNA. The details and mechanisms of this process are still unknown. While exploring the stage of poliovirus functioning, we studied the interactions of the cytoplasmic form of human glycyl-tRNA synthetase and its domains with fragments of the poliovirus IRES element. As a result, we identified the minimal fragment of the viral mRNA that glycyl-tRNA synthetase adequately interacts with and we estimated the contribution of some of the domains to the interaction between glycyl-tRNA synthetase and RNA.     

Genome-Wide Analysis of Biotin Biosynthesis in Eukaryotic Photosynthetic Algae

Biotin is a cofactor responsible for carbon dioxide transfer in several carboxylase enzymes, which play a significant role in various metabolic reactions such as fatty acid synthesis, branched chain amino acid catabolism, and gluconeogenesis. Biotin is also involved in citric acid cycle, which is the process of biochemical energy generation during aerobic respiration. Though the function of biotin in the growth of algae has been extensively investigated, little is known about the biosynthetic routes of biotin in the algal kingdom. In the present study, 44 biotin biosynthesis-related genes were identified from 14 eukaryotic photosynthetic algal genomes by BLASTP and TBLASN programs. A comprehensive analysis was performed to characterize distribution, phylogeny, structure domains, and coevolution patterns of those genes. Forty-four biotin biosynthesis-related enzymes (BBREs) were found to be distributed in three groups: 7-keto-8-aminopelargonic acid synthase, diaminopelargonic acid synthase/dethiobiotin synthetase, and biotin synthase. Structure domains were considerably conserved among the subfamilies of BBREs. The intramolecular coevolutionary sites are widely distributed in biotin synthase. The present study provides new insights into the origin and evolution of biotin biosynthetic pathways in eukaryotic photosynthetic algae. Furthermore, the characterization of biotin biosynthesis-related genes from algae will promote the identification and functional studies of BBREs.     

Crystal structure of carbapenam synthetase (CarA)

Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics.     

Human long-chain acyl-CoA synthetase: structure and chromosomal location.

A complementary DNA clone encoding the entire human long-chain acyl-CoA synthetase was isolated and the total 698-amino acid sequence was deduced. The amino acid sequence of human long-chain acyl-CoA synthetase shows 84.9% identity to that of rat long-chain acyl-CoA synthetase. The nucleotide sequences of the protein coding regions between human and rat long-chain acyl-CoA synthetase mRNAs are highly conserved (85.6%), whereas those of the 3' untranslated regions are less conserved (72%). The location of the human long-chain acyl-CoA synthetase gene was identified on chromosome 4 by spot hybridization of flow-sorted chromosomes. Computer-assisted homology search revealed a significant similarity of the enzyme with the enzymes of the luciferase family. Based on this similarity, the structure of human long-chain acyl-CoA synthetase can be divided into five domains: the N-terminus, two domains similar to those in enzymes of the luciferase family, a long gap region between the similar domains and the C-terminus.     

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.     

Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity

Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products. Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase. Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V. harveyi fatty acid synthase (but not acyl-ACP synthetase) activity. In contrast, various replacements of Ala-45 were tolerated by both enzymes. None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis. These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.     

Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase

Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with l-aspartate oxidase, the other component of the so-called "quinolinate synthetase complex," the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken. The results are presented in this paper. Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up. The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E. coli culture. A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented. Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from l-aspartate, dihydroxyacetone phosphate, and O(2) by using only nadA and nadB gene overexpressed products.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.