Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis

The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.     

NovJ/NovK catalyze benzylic oxidation of a beta-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis

The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating beta-OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J2K2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the beta-OH of the tyrosyl moiety to yield the tethered beta-ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile beta-ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity.     

Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein

Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations. The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.     

Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase

The excised C-terminal thioesterase (TE) domain from the multidomain tyrocidine nonribosomal peptide synthetase (NRPS) was recently shown to catalyze head-to-tail cyclization of a decapeptide thioester to form the cyclic decapeptide antibiotic tyrocidine A [Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A., and Walsh, C. T. (2000) Nature 407, 215-218]. The peptide thioester substrate was a mimic of the TE domain's natural, synthetase-bound substrate. We report here the synthesis of modified peptide thioester substrates in which parts of the peptide backbone are altered either by the replacement of three amino acid blocks with a flexible spacer or by replacement of individual amide bonds with ester bonds. Rates of TE domain catalyzed cyclization were determined for these substrates and compared with that of the wild-type substrate, revealing that some parts of the peptide backbone are important for cyclization, while other parts can be modified without significantly affecting the cyclization rate. We also report the synthesis of a modified substrate in which the N-terminal amino group of the wild-type substrate, which is the nucleophile in the cyclization reaction, is replaced with a hydroxyl group and show that this compound is cyclized by the TE domain to form a macrolactone at a rate comparable to that of the wild-type substrate. These results demonstrate that the TE domain from the tyrocidine NRPS can catalyze cyclization of depsipeptides and other backbone-substituted peptides and suggest that during the cyclization reaction the peptide substrate is preorganized for cyclization in the enzyme active site in part by intramolecular backbone hydrogen bonds analogous to those in the product tyrocidine A.     

Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa

The thiazoline-containing siderophores pyochelin, yersiniabactin, and Micacocidin A all have D-thiazoline rings, participating in high-affinity chelation of ferric iron. However, studies with pyochelin (Pch) synthetase and yersiniabactin (Ybt) synthetase reconstituted from pure protein components have shown that only L-cysteine is activated and tethered as a covalent aminoacyl-S-enzyme intermediate. Nor are any of the canonical epimerase domains of nonribosomal peptide synthetase (NRPS) assembly lines found in the Ybt or Pch synthetase modules. Here, we report that the PchE subunit of the Pch synthetase exchanges solvent deuterium into the C(2) center of the thiazoline moieties during siderophore chain elongation. Both PchE and HMWP2, from Ybt synthetase, subunits have a 310-360-residue insert in their amino acid activation domains that look like defective methyltransferase (MT) domains. We suggest these inserts are noncanonical epimerase domains, reversibly deprotonating and reprotonating acyl-S-enzyme intermediates at the C(2) locus. The PchE subunit does not epimerize the Cys-S-enzyme intermediate, but once amide bond formation from a benzoyl-S-PchE donor is catalyzed by the cyclization (Cy) domain of PchE, the N-benzoyl-Cys-S-PchE intermediate is present as a D,L-mixture. The subsequent phenylthiazolinyl-S-PchE intermediate, arising from cyclodehydration of the N-benzoyl-Cys-S-PchE intermediate, is likewise a D,L-mixture on hydrolytic release and enantiomer analysis. These results suggest a default role for MT domains of NRPS assembly lines in generating alpha-carbanionic species from thioester intermediates during siderophore chain elongation.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of cyclic peptides from unprotected precursors using removable N alpha-(1-(4-methoxyphenyl)-2-mercaptoethyl) auxiliary.

A new method to cyclize unprotected peptides is presented. The method involves the use of a 1-phenyl-2-mercaptoethyl derivative on the N-terminal glycine. This template acts as an auxiliary thiol-containing group in order to drive cyclization with a counterpart thioester moiety on the same molecule. Subsequent facile removal of the derivative generates products with only native peptide structure. The successful, high-yield cyclization of the peptide GSPYSSDTTPA is described.     

Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase

Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and 13C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is approximately 3. The 13C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. The intrinsic alpha-secondary deuterium isotope effect at C-4 of the nicotinamide ring of NAD is approximately 1.4; this large normal value (the equilibrium isotope effect is 0.89) shows tight coupling of hydrogen motions in the transition state accompanied by tunneling. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is approximately 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed 13C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the 13C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the 13C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein.

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH(2)-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.     

Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro reconstitution of aryl-4, 2-bisthiazoline synthetase activity from PchD, PchE, and PchF.

Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribosomal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expressed in and purified from Escherichia coli and are found to produce the tricyclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores. The three proteins contain three adenylation domains, one specific for salicylate activation and two specific for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming with phosphopantetheine to enable covalent tethering of salicyl and cysteinyl moieties as acyl-S-enzyme intermediates. Two cyclization domains (Cy1 in PchE and Cy2 in PchF) create the two amide linkages in the elongating chains and the cyclodehydrations of acylcysteine moieties into thiazolinyl rings. The ninth domain, the most downstream domain in PchF, is the chain-terminating, acyl-S-enzyme thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product. A PchF-thioesterase domain active site double mutant fails to turn over, but a monocyclic hydroxyphenyl-thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.     

Identification and characterization of the pswP gene required for the parallel production of prodigiosin and serrawettin W1 in Serratia marcescens

Serratia marcescens mutants defective in production of the red pigment prodigiosin and the biosurfactant serrawettin W1 in parallel were isolated by transposon mutagenesis of strain 274. Cloning of the DNA fragment required for production of these secondary metabolites with different chemical structures pointed out a novel open reading frame (ORF) named pswP. The putative product PswP (230 aa) has the distinct signature sequence consensus among members of phosphopantetheinyl transferase (PPTase) which phosphopantetheinylates peptidyl carrier protein (PCP) mostly integrated in the nonribosomal peptide synthetases (NRPSs) system. Since serrawettin W1 belongs to the cyclodepsipeptides, which are biosynthesized through the NRPSs system, and one pyrrole ring in prodigiosin has been reported as a derivative of L -proline tethered to phosphopantetheinylated PCP, the mutation in the single gene pswP seems responsible for parallel failure in production of prodigiosin and serrawettin W1.     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Tandem heterocyclization activity of the multidomain 230 kDa HMWP2 subunit of Yersinia pestis yersiniabactin synthetase: interaction of the 1-1382 and 1383-2035 fragments.

The six-domain, 2035-amino acid subunit high-molecular weight protein 2 (HMWP2) activates salicylate and two cysteines and loads them covalently on its three carrier protein domains during assembly of the iron-chelating virulence factor, yersiniabactin of the plague bacterium Yersinia pestis. The 1-1382 fragment of HMWP2 (ArCP-Cy1-A), overproduced in Escherichia coli, contains the first three domains: the aryl carrier protein (ArCP) domain, the cysteine specific adenylation domain (A), and the first condensation/cyclization domain (Cy1). The ArCP can be posttranslationally phosphopantetheinylated on Ser52 and then loaded with a salicyl group on the phosphopantetheine (Ppant) thiol by action of the YbtE, a salicyl-AMP ligase. The HMWP2 1-1382 fragment can activate L-cysteine as Cys-AMP. The HMWP2 1383-2035 fragment contains the remaining three domains: two peptidyl carrier proteins (PCP1 and PCP2) separated by a second condensation/cyclization domain (Cy2). Phosphopantetheinylation of the HMWP2 1383-2035 fragment at Ser1439 (PCP1) and Ser1977 (PCP2) facilitates cysteinylation of both thiols by HMWP2 1-1382. When the holo 1-1382 and bis-holo 1383-2035 protein fragments are mixed with ATP, salicylate, and cysteine, four products are slowly released [salicylcysteine (Sal-Cys), (hydroxyphenylthiazolinyl)cysteine (HPT-Cys), HPT-Cys-Cys, and the bisheterocyclic HPTT-Cys], reflecting thiolytic rerouting by cysteine in solution of elongating acyl-S-enzyme intermediates tethered at ArCP, PCP1, and PCP2 carrier protein domains, respectively. Conducting the in trans reconstitution with the S1439A mutant of HMWP2 1383-2035 releases only Sal-Cys, while the S1977A mutant leads to HPT-Cys formation but not HPT-Cys-Cys or HPTT-Cys. These results suggest localization of particular acyl-S-enzyme intermediates to each of the three carrier protein regions and also establish the sequential action of Cy1 and Cy2, with the latter producing the tandem 4,2-bisheterocyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) moiety characteristic of this class of siderophores.     

Methionine metabolism and ethylene biosynthesis in senescing petals of Tradescantia

The endogenous content of methionine in isolated petals of Tradescantia was found to increase during petal senescence while the levels of S-methylmethionine and protein were found to decline. The increase in free methionine was, at least in part, the result of protein degradation. Methionine and homocysteine were shown to be intermediates in ethylene biosynthesis while S-methylmethionine was not involved. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to all floral tissues resulted in large stimulations of ethylene production. ACC was shown to be an endogenous amino acid the internal levels of which correlated positively with the rate of ethylene production. Application of l-methionine-[U-¹⁴C] led to a rapid appearance of radioactivity in both ethylene and ACC. The specific radioactivity of C-2 and C-3 of ACC and that of ethylene were found to be nearly identical which indicated that ACC was the immediate precursor of ethylene in senescing petals of Tradescantia.     

Overexpression of Acetyl-CoA Carboxylase Gene of <Emphasis Type="Italic">Mucor rouxii</Emphasis> Enhanced Fatty Acid Content in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.     

The interconversion of ACC deaminase and <Emphasis Type="SmallCaps">d</Emphasis>-cysteine desulfhydrase by directed mutagenesis

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.     

The Burkholderia cenocepacia BDSF quorum sensing fatty acid is synthesized by a bifunctional crotonase homologue having both dehydratase and thioesterase activities

Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis-2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis-2-dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis-2-dodecenoyl-ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation pathway. We demonstrated Bcam0581to be a bifunctional protein that not only catalysed dehydration of 3-hydroxydodecanoyl-ACP to cis-2-dodecenoyl-ACP, but also cleaved the thioester bond to give the free acid. Both activities required the same set of active-site residues. Although dehydratase and thioesterase activities are known activities of the crotonase superfamily, Bcam0581 is the first protein shown to have both activities.