Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains

The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain. VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.     

Catalytic mapping of the vibriobactin biosynthetic enzyme VibF.

The iron-chelating catechol siderophore vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit, ten-domain nonribosomal peptide synthetase system, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two (dihydroxyphenyl)methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have utilized site-specific and domain-deletion mutagenesis to map the heterocyclization and primary and secondary amine acylation activities of the six-domain (Cy1-Cy2-A-C1-PCP-C2) VibF subunit. We have found that Cy2 is capable of and limited to the condensation (amide bond formation) step of the three-step heterocyclization process, while Cy1 is capable of and limited to the final processing (cyclization/dehydration) steps to the completed heterocycle. Additionally, we have observed that the C2 domain functions in both N(9) (primary amine) acylation and N(5) (secondary amine) acylation of the (dihydroxybenzoyl)norspermidine substrate, leaving no catalytic role for the C1 domain, a conclusion confirmed with the formation of vibriobactin in a C1-deficient system. Thus VibF is an NRPS with two domains, Cy1 and Cy2, that perform a function otherwise performed by one and with one domain, C2, that performs a function otherwise performed by two. While C2 appeared to tolerate uncyclized threonine in place of the usual heterocycle in primary amine acylation, it refused this replacement in the corresponding donor substrate in secondary amine acylation.     

Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.     

Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate

The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.     

Carrier protein recognition in siderophore-producing nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.     

Catalytically inactive condensation domain C1 is responsible for the dimerization of the VibF subunit of vibriobactin synthetase

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide synthases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. The oligomeric structure of these assembly line enzymes has been a topic of interest because higher order oligomeric quaternary structural arrangements allow for alternate paths of acyl intermediate elongation and present unique challenges for the chimeric engineering of hybrid assembly lines. Unlike other NRPS systems that in general appear to be monomeric, the six domain (Cy1-Cy2-A-C1-PCP-C2) VibF subunit of vibriobactin synthetase has previously been shown to be dimeric, the same oligomeric state as that observed for FAS and PKS assembly lines. It has been demonstrated that the C1 domain within VibF is catalytically inactive and is not required for vibriobactin production. Utilizing sedimentation equilibrium analytical ultracentrifugation experiments to determine the oligomeric states of several VibF subfragments, we report that the C1 domain is largely responsible for VibF dimerization. Comparative rates of vibriobactin production, coupled with dissociation constants for VibF subfragment pair heterocomplexes, suggest that the mere presence of C1 does not detectably enhance the catalytic rates of neighboring domains, but it may properly orient Cy1-Cy2-A relative to PCP-C2.     

Vibriobactin, a siderophore from Vibrio cholerae

A novel siderophore (microbial iron transport compound) has been isolated from low iron cultures of Vibrio cholerae. Belonging to the catecholamide family of chelators, it has been shown to contain three residues of 2,3-dihydroxybenzoic acid and two residues of threonine. Both threonine moieties are present in the form of oxazoline rings. Furthermore, the polyamine backbone of the molecule was proved to be not spermidine, but the rare N-(3-aminopropyl)-1,3-diaminopropane, norspermidine. The structure of the new siderophore has been determined to be N-[3-(2,3-dihydroxybenzamido)propyl]-1, 3-bis[2,3-dihydroxyphenyl)-trans-5-methyl-2-oxazoline-4-carboxamido]prop ane. The compound has been given the trivial name vibriobactin. Mutants defective in the synthesis and utilization of vibriobactin were isolated. In an iron-limited environment V. cholerae was found to respond more strongly to vibriobactin, agrobactin, and ferrichrome than to enterobactin.     

The structure of VibH represents nonribosomal peptide synthetase condensation,cyclization and epimerization domains

Nonribosomal peptide synthetases (NRPSs) are large, multidomain enzymes that biosynthesize medically important natural products. We report the crystal structure of the free-standing NRPS condensation (C) domain VibH, which catalyzes amide bond formation in the synthesis of vibriobactin, a Vibrio cholerae siderophore. Despite low sequence identity, NRPS condensation enzymes are structurally related to chloramphenicol acetyltransferase (CAT) and dihydrolipoamide acyltransferases. However, although the latter enzymes are homotrimers, VibH is a monomeric pseudodimer. The VibH structure is representative of both NRPS condensation and epimerization domains, as well as the condensation-variant cyclization domains, which are all expected to be monomers. Surprisingly, despite favorable positioning in the active site, a universally conserved histidine important in CAT and in other C domains is not critical for general base catalysis in VibH.     

Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis

Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.     

Thioesterase portability and peptidyl carrier protein swapping in yersiniabactin synthetase from Yersinia pestis

Multimodular enzymes, including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and mixed PKS/NRPS systems, contain functional domains with similar functions. Domain swapping and module fusion are potential powerful strategies for creating hybrid enzymes to synthesize modified natural products. To explore these strategies, yersiniabactin (Ybt) synthetase containing two subunits, HMWP2 [two NRPS modules (N-terminus-ArCP-Cy1-A-PCP1 and Cy2-PCP2-C-terminus)] and HMWP1 [one PKS (N-terminus-KS-AT-MT1-KR-ACP) one NRPS module (Cy3-MT2-PCP3-TE-C-terminus)], was used as a model system to study peptidyl carrier protein (PCP) domain swapping, thioesterase (TE) portability, and module-module fusion. The PCP1 domain of the N-terminal NRPS module of HMWP2 was swapped with either PCP2 or PCP3. The fusion proteins were 3-8-fold less active than the wild-type protein. The swapping of PCP2 of HMWP2 abolished the heterocyclization activity of the Cy2 domain while retaining its condensation function. When the two PCPs of HMWP2 were swapped by PCP3TE, it created two active fusion proteins: one or two NRPS modules fused to the TE domain. The internal TE domain of the two fusion proteins catalyzed the hydrolysis of enzyme-bound intermediates HPT-S-PCP3 to form HPT-COOH and HPTT-S-PCP3 to form HPTT-COOH. The TE activity was eliminated by the S2980A point mutation at its active site. Therefore, the three PCPs of the Ybt synthetase were swappable, and its lone TE domain was portable. The reasons for the observed low activities of the fusion proteins and lessons for protein engineering in generating novel modular enzymes were discussed.     

肽基载体蛋白结构功能的研究进展

肽基载体蛋白(peptidyl carrier protein,PCP)是非核糖体肽合成酶(non-ribosomal peptide synthetase,NRPS)的核心结构域。根据NRPS的装配机制,每个模块都至少包含一个PCP,PCP对于非核糖体肽合成中氨基酸残基及多肽在不同催化结构域中的传递起着重要作用,并为氨基酸残基和多肽向模块内其他修饰酶的转移提供一个平台。本文主要对PCP的结构功能、与其他催化结构域的相互作用及重组模块活性降低的问题等方面进行了综述,期望为重组NRPS模块的构建提供理论依据。     

Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.     

Structure of vulnibactin,a new polyamine-containing siderophore from Vibrio vulnificus

A new siderophore named vulnibactin has been isolated from low iron cultures of Vibrio vulnificus, a human pathogen. The structure was established as N-[3-(2,3-dihydroxybenzamido)propyl]-1,3-bis[2-(2-hydroxyphenyl)-trans-5-methyl-2-oxazoline-4-carboxamido]propane by a combination of acid hydrolysis, nuclear magnetic resonance spectroscopy and positive fast atom bombardment mass spectrometry. Vulnibactin is characterized as containing one residue of 2,3-dihydroxybenzoic acid as well as two residues of salicylic acid, both of which are involved in the formation of oxazoline rings with l-threonine bound to a norspermidine backbone. In addition, two other compounds with siderophore activity were purified and their structures were also determined. These two compounds provided further support for the structure of vulnibactin.     

Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi

A catechcol-type siderophore, assigned the trivial name chrysobactin, was isolated from the phytopathogenic bacterium Erwinia chrysanthemi and characterized by degradation and spectroscopic techniques as N-[N2-(2,3-dihydroxybenzoyl)-D-lysyl]-L-serine. Chrysobactin, which was also obtained by chemical synthesis, was shown to be active in supplying iron to a group of mutants of E. chrysanthemi defective in biosynthesis of the siderophore.     

Ebony,a novel nonribosomal peptide synthetase for beta-alanine conjugation with biogenic amines in Drosophila

Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, we provide evidence for its substrate specificity and reaction mechanism. Ebony activates beta-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on beta-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, we define Ebony as a beta-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. We propose that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through beta-alanyl conjugation.     

Analyses of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis

The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N(6) acylation of the central l-Lys residue and subsequent N(6)-hydroxylation of the central N(6)-acyl-l-Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N(6)-acyl-N(6)-hydroxy-l-Lys and N(6)-hydroxy-l-Lys, respectively. Furthermore, the preference of the A domain of MbtB for l-Thr over l-Ser suggests that the megasynthase produces MBT derivatives with β-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this β-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.     

Synthesis of an encapsulated arsenic anion via reaction of arsenic acid with a linear catecholamide-N,N′,N″-tris(2,3-dihydroxybenzoyl)-1,5,10-triazadecane

The reaction of arsenic acid with N,N′,N″-Tris(2,3-dihydroxybenzoyl)-1,5,10-Triazadecane(3,4-LICAM) provides the first example of an encapsulated arsenic anion. The structural features of this compound, thought to have an octahedral configuration around the arsenic, was established by ¹³C NMR, IR, UV and FAB mass spectroscopy as well as elemental analysis.     

Selectivity of the yersiniabactin synthetase adenylation domain in the two-step process of amino acid activation and transfer to a holo-carrier protein domain

The adenylation (A) domain of the Yersinia pestis nonribosomal peptide synthetase that biosynthesizes the siderophore yersiniabactin (Ybt) activates three molecules of L-cysteine and covalently aminoacylates the phosphopantetheinyl (P-pant) thiols on three peptidyl carrier protein (PCP) domains embedded in the two synthetase subunits, two in cis (PCP1, PCP2) in subunit HMWP2 and one in trans (PCP3) in subunit HMWP1. This two-step process of activation and loading by the A domain is analogous to the operation of the aminoacyl-tRNA synthetases in ribosomal peptide synthesis. Adenylation domain specificity for the first step of reversible aminoacyl adenylate formation was assessed with the amino acid-dependent [(32)P]-PP(i)-ATP exchange assay to show that S-2-aminobutyrate and beta-chloro-L-alanine were alternate substrates. The second step of A domain catalysis, capture of the bound aminoacyl adenylate by the P-pant-SH of the PCP domains, was assayed both by catalytic release of PP(i) and by covalent aminoacylation of radiolabeled substrates on either the PCP1 fragment of HMWP2 or the PCP3-thioesterase double domain fragment of HMWP1. There was little selectivity for capture of each of the three adenylates by PCP3 in the second step, arguing against any hydrolytic proofreading of incorrect substrates by the A domain. The holo-PCP3 domain accelerated PP(i) release and catalytic turnover by 100-200-fold over the leak rate (<1 min(-1)) of aminoacyl adenylates into solution while PCP1 in trans had only about a 5-fold effect. Free pantetheine could capture cysteinyl adenylate with a 25-50-fold increase in k(cat) while CoA was 10-fold less effective. The K(m) of free pantetheine (30-50 mM) was 3 orders of magnitude larger than that of PCP3-TE (10-25 microM), indicating a net 10(4) greater catalytic efficiency for transfer to the P-pant arm of PCP3 by the Ybt synthetase A domain, relative to P-pant alone.     

Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus

We isolated a novel gram-positive bacterium, Brevibacillus texasporus, that produces an antibiotic, BT. BT is a group of related peptides that are produced by B. texasporus cells in response to nutrient limitation. We report here purification and determination of the structure of the most abundant BT isomer, BT1583. Amino acid composition and tandem mass spectrometry experiments yielded a partial BT1583 structure. The presence of ornithine and d-form residues in the partial BT1583 structure indicated that the peptide is synthesized by a nonribosomal peptide synthetase (NRPS). The BT NRPS operon was rapidly and accurately identified by using a novel in silico NRPS operon hunting strategy that involved direct shotgun genomic sequencing rather than the unreliable cosmid library hybridization scheme. Sequence analysis of the BT NRPS operon indicated that it encodes a colinear modular NRPS with a strict correlation between the NRPS modules and the amino acid residues in the peptide. The colinear nature of the BT NRPS enabled us to utilize the genomic information to refine the BT1583 peptide sequence to Me(2)-4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH2OH. In addition, we report the discovery of novel NRPS codons (sets of the substrate specificity-conferring residues in NRPS modules) for valine, lysine, ornithine, and tyrosine.     

Synthesis of N3-fumaramoyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase

Several analogues of N3-fumaramoyl-L-2,3-diaminopropanoic acid were synthesized and evaluated for inhibition of glucosamine-6-phosphate synthetase activity. The syntheses were accomplished by acylation reaction of N2-tert.-butoxycarbonyl-L-2,3-diaminopropanoic acid (Boc-A2pr) or N2-tert.-butoxycarbonyl-L-2,4-diaminobutanoic acid (Boc-A2-bu) with the N-succinimidoyl esters of several derivatives of alpha, beta-unsaturated acids 2a-d followed by deprotection of the Boc groups. The obtained compounds were tested for inhibition of glucosamine synthetase isolated from Salmonella typhimurium and Saccharomyces cerevisiae. The results indicated that among the synthesized compounds, N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP) was the most powerful inhibitor of glucosamine synthetase.