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Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved Bacillus subtilis cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.

The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5ʹ-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis. [ABSTRACT FROM AUTHOR]

The incorporation of non-proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20-22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion-protein-based design for synthetic tRNA-aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA-binding domain (Arc1p-C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p-C using flexible linkers and achieved tRNA-aminoacylation with both proteinogenic and non-proteinogenic amino acids. The resulting aminoacyl-tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA-aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non-proteinogenic amino acids. [ABSTRACT FROM AUTHOR]

The Bacillus subtilis laboratory strain JH642 shows a cold-sensitive phenotype after a temperature shift from 37 to 15°C in comparison to wild type strain MR168. A mutation in the acetolactate synthase complex IlvBH was found to be partially responsible for this growth defect after cold shock. Via DNA sequencing, genetic and biochemical studies, this defect was characterized, which entails a substitution of two adenines to guanines in the ilvB gene. This results in an amino acid substitution from lysine at position 176 to glycine. As a consequence, the acetolactate synthase efficiency in strain JH642 was found to be reduced by 51-fold. [ABSTRACT FROM AUTHOR]

Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set of core domains with all essential catalytic functions necessary for the incorporation and modification of one building block. Very often, d-amino acids are found in such products which, with few exceptions, are generated by the action of NRPS integrated epimerization (E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein (PCP) bound l-intermediate. In this study we present a quantitative investigation of substrate specificity of four different E domains (two ‘peptidyl-’ and two ‘aminoacyl-’E domains) derived from different NRPSs towards PCP bound peptides. The respective PCP-E bidomain apo-proteins (TycB3-, FenD2-, TycA- and GrsA-PCP-E) were primed with various peptidyl-CoA precursors by utilizing the promiscuous phosphopantetheinyl transferase Sfp. PCP bound peptidyl- S-Ppant epimerization products were chemically cleaved and analyzed for their l/ d-ratios by LCMS. We were able to show that all four E domains tolerate a broad variety of peptidyl- S-Ppant-substrates as evaluated by kobs values and final l/ d-product equilibria determined for each reaction. The two C-terminal amino acids of the substrate seem to be recognized by ‘peptidyl-’E domains. Interestingly, the ‘aminoacyl-’E domains GrsA- and TycA-E were also able to convert the elongated intermediates. All four E domains accepted an N-methylated precursor as well and epimerized this substrate with high efficiency. Finally, we could demonstrate that the condensation (C) domain of TycB1 is also able to process peptidyl substrates transferred by TycA. In conclusion, these findings are of great impact on future engineering attempts. [ABSTRACT FROM AUTHOR]

The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4′-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 Å resolution reveals a novel α/β-fold exhibiting an unexpected intra-molecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homo-dimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate. [ABSTRACT FROM AUTHOR]

An extranuclear oligomycin-resistant mutant of Aspergillus nidulans was isolated and biochemically analyzed. The mutant grows slowly in the absence of oligomycin and contains an excess of cytochrome c. The ATPase complex solubilized from mutant mitochondria with Triton X-100 is 80 times more resistant to oligomycin than the enzyme of the parental strain. The enzyme complexes of both strains were further purified to electrophoretic homogeneity and resolved by dodecylsulfate gel electrophoresis in the presence of 8 M urea into 16 polypeptide bands of apparent molecular weights between 65000 and 6000. Four proteins of apparent molecular weights 43000, 21000, 11000 and 6000 are synthesized on cycloheximide-resistant mitochondrial ribosomes. The smallest subunit is selectively extracted from the purified enzyme by neutral chloroform/methanol (2/1). A proteolipid of identical electrophoretic mobility and site of synthesis is the major component of proteins extractable with the same solvent from whole mitochondrial membranes. This protein has been isolated from the mutant and its parental strain, and purified to apparent homogeneity. The proteins from both strains start with N-formyl-methionine and end with valine. However, a second internal methionine residue present in the wild-type protein is absent, together with some other amino acids, from the mutant protein. It is concluded that this small lipophilic protein is coded by a mitochondrial gene determining oligomycin resistance, is synthesized on mitochondrial ribosomes and is associated with the membrane sector of the ATPase complex. The mutational alteration of the protein not only confers oligomycin resistance to the ATPase complex but also causes a structural alteration of the mitochondrial inner membrane. [ABSTRACT FROM AUTHOR]

The major cold-shock protein of Bacillus subtilis, CspB, is a member of a protein family widespread among prokaryotes and eukaryotes that share the highly conserved cold-shock domain (CSD). The CSD domain is involved in transcriptional and translational regulation and was shown to bind the Y-box motif, a cis-element that contains the core sequence ATTGG, with high affinity. The three-dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD domain creates a surface rich in aromatic and basic amino acids that may act as the nucleic acid-binding site. We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site-directed mutagenesis. In gel retardation and ultra-violet cross-linking experiments, the ability of CspB mutants to bind single-stranded oligonucleotides (ssDNA) that contain the Y-box motif was investigated. Single substitutions of three highly conserved phenylalanine residues (Phe-15, Phe-17, Phe-27) by alanine and substitution of one histidine (His-29) by glutamine, all located within the putative RNA-binding sites RNP-1 and RNP-2, abolished the nucleic acid-binding activity of CspB. Conservative substitutions of Phe-15 to tyrosine (F15Y) showed a small increase in binding affinity, whereas separate replacement of Phe-17 and Phe-27 by tyrosine caused a reduction in binding activity. These and other substitutions including the conserved basic residues Lys-7, Lys-13 and Arg-56 as well as the aromatic residues Trp-8 and Phe-30 strongly suggest that CspB uses the side-chains of these amino acids for specific interaction with nucleic acids. Ultraviolet cross-linking experiments for CspB mutants with ssDNA supported the idea of specific CspB/nucleic acid interaction and indicated an essential role for the aromatic and basic residues in this binding. In addition, two-dimensional nuclear magnetic resonance studies with F17A, K13Q, F15Y and F27Y revealed... [ABSTRACT FROM AUTHOR]

Two major families of peptidylprolyl cis- trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative ( ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246° on the genetic map upstream of the clpX and lonA, B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity ( kcat/ Km of 1.5 μM−1 s−1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor. [ABSTRACT FROM AUTHOR]

The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of ∼60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 Å resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94° occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the α-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation. [ABSTRACT FROM AUTHOR]

4'-Phosphopantetheinyl transferases (PPTases) are essential for the production of fatty acids by fatty acid synthases (primary metabolism) and natural products by nonribosomal peptide synthetases and polyketide synthases (secondary metabolism). These systems contain carrier proteins (CPs) for the covalent binding of reaction intermediates during synthesis. PPTases transfer the 4'-phosphopantetheine moiety from coenzyme A (C0A) onto conserved serine residues of the apo-CPs to convert them to their functionally active holo form. In bacteria, two types of PPTases exist that are evolutionary related but differ in their substrate spectrum. Acyl carrier protein synthases (AcpSs) recognize CPs from primary metabolism, whereas Sfp- (surfactin production-) type PPTases have a preference for CPs of secondary metabolism. Previous investigations showed that a peptidyl carrier protein (PCP) of secondary metabolism can be altered to serve as substrate for AcpS. We demonstrate here that a single mutation in PCP suffices for the modification of this CP by AcpS, and we have identified by mutational analysis several other PCP residues and two AcpS residues involved in substrate discrimination by this PPTase. These altered PCPs were still capable of serving their designated function in NRPS modules, and selective use of AcpS or Sfp leads to production of two different products by a trimodular NRPS. [ABSTRACT FROM AUTHOR]

Here, we report a new experimental approach utilizing an amide ligation reaction for the characterization of acyl carrier protein (ACP)-bound reaction intermediates, which are otherwise difficult to analyze by traditional biochemical methods. To explore fatty acid tailoring enzymes of the calcium-dependent antibiotic (CDA) biosynthetic pathway, this strategy enabled the transformation of modified fatty acids, covalently bound as thioesters to an ACP, into amide ligation products that can be directly analyzed and compared to synthetic standards by HPLC-MS. The driving force of the amide formation is the thermodynamic activation inherent to thioester-bound compounds. Using this novel method, we were able to characterize the ACP-mediated biosynthesis of the unique 2,3-epoxyhexanoyl moiety of CDA, revealing a new type of FAD-dependent oxidase HxcO with intrinsic enoyl-ACP epoxidase activity, as well as a second enoyl-ACP epoxidase, HcmO. In general, our approach should be widely applicable for the in vitro characterization of other biosynthetic systems acting on carrier proteins, such as integrated enzymes from NRPS and PKS assembly lines or tailoring enzymes of fatty and amino acid precursor synthesis. [ABSTRACT FROM AUTHOR]

The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues Dptl from the daptomycin producer Streptomyces roseosporus and Lptl from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, Dptl, and Lptl are S-adenosyl methionine (SAM)-dependent α-ketoglutarate methyltransferases that catalyze the stereospecific methylation of α-ketoglutarate (αKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase lIvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of αKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of β-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes. [ABSTRACT FROM AUTHOR]

The linear pentadecapeptide gramicidin has been reported to be assembled by four large multimodular nonribosomal peptide synthetases (NRPSs), LgrABCD, that comprise 16 modules. During biosynthesis, the N-formylated 16mer peptide is bound to the peptidyl carrier protein (PCP) of the terminal module via a thioester bond to the carboxyl group of the last amino acid glycine16. In a first reaction the peptide is released from the protein template in an NAD(P)H-dependent reduction step catalyzed by the adjacent reductase forming an aldehyde intermediate. Here we present the biochemical proof that this aldehyde intermediate is further reduced by an aldoreductase, LgrE, in an NADPH-dependent manner to form the final product gramicidin A, N-formyl-pentadecapeptide-ethanolamine. To determine the potential use of the two reductases in the construction of hybrid NRPSs, we have tested their ability to accept a variety of different substrates in vitro. The results obtained give way to a broad spectrum of possible use. [ABSTRACT FROM AUTHOR]

The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of poluketide synthases (PKSs), and fatty acid sythases (FASs) totheir active holo form is accomplished with dedicated 4'-phosphopantethenyl transferases (PPTases). They catalyze the transfers of the essential prosthetic group 4'-phosphopantethennyl (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs. PPTases, based on sequence and substrate specifity, have beeb classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism. The recently solved crystal structures of AcpS and Sfp-type PPTase with CoA revealed a common α + β fold with a β1α 3β2 motif and similarities in CoA binding and polymerization mode. However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal. In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP. That, in turn, acts as a nucleophile to attack the β-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between β4 and α5 (residues T111-S124) in Sfp is the PCP binding region. Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structural to coordinate CoA are now all confirmed by mutational and biochemical and biochemical analysis. [ABSTRACT FROM AUTHOR]

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating L- and D-configuration forming a β-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp. Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptideethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro. [ABSTRACT FROM AUTHOR]