Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism

The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases). They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (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 been 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 PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) 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 beta-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (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 structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.     

4'-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis

4'-Phosphopantetheine transferases (PPTases) transfer the 4'-phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases. This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form. We have investigated the 4'-phosphopantetheinylation reaction in Bacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcB encoding AcpS from B. subtilis, which complemented an Escherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically characterized. AcpS also modified the d-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested. We thereby extend the reported broad specificity of this enzyme to the homologous ACP. This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B. subtilis sfp(+) strain. The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.     

Characterization of a nonspecific phosphopantetheinyl transferase from Pseudomonas syringae pv. syringae FF5

The 4'-phosphopantetheinyl transferases (PPTases) catalyze the transfer of a 4'-phosphopantetheine moiety from coenzyme A to phosphopantetheine-dependent carrier proteins. The carrier proteins (CPs) are required for the biosynthesis of peptides synthesized by nonribosomal peptide synthases and the biosynthesis of fatty acids and polyketides. A single PPTase (PcpS) is present in the pathogenic bacterium Pseudomonas aeruginosa. Several pathovars of Pseudomonas syringae produce the chlorosis-inducing phytotoxin coronatine. Structural genes for coronatine biosynthesis include two ACPs, two ACP domains, and one peptidyl carrier protein (PCP) domain. To gain insight into factors affecting coronatine biosynthesis, the PPTase of P. syringae pv. syringae FF5 has been investigated. A single PPTase gene (pspT) was amplified from this organism by PCR. The translation product PspT exhibited 62% identity to PcpS as well as higher levels of identity to other, uncharacterized Pseudomonad PPTases. PspT was overproduced in soluble form in Escherichia coli and its enzymatic properties were compared with those of PcpS. PspT exhibited broad substrate specificity, and it displayed the highest activity with a PCP domain. In contrast, the most efficient substrates for PcpS are CPs from primary metabolism. These results indicate phosphopantetheinyl transferases from different Pseudomonas sp. may vary significantly in their enzymatic properties.     

Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp

The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.     

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.     

Gene cloning,expression and functional characterization of a phosphopantetheinyl transferase from <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> serotype O1

Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.     

Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa

Phosphopantetheinyl-dependent carrier proteins are part of fatty-acid synthases (primary metabolism), polyketide synthases, and non-ribosomal peptide synthetases (secondary metabolism). For these proteins to become functionally active, they need to be primed with the 4'-phosphopantetheine moiety of coenzyme A by a dedicated phosphopantetheine transferase (PPTase). Most organisms that employ more than one phosphopantetheinyl-dependent pathway also have more than one PPTase. Typically, one of these PPTases is optimized for the modification of carrier proteins of primary metabolism and rejects those of secondary metabolism (AcpS-type PPTases), whereas the other, Sfp-type PPTase, efficiently modifies carrier proteins involved in secondary metabolism. We present here a new type of PPTase, the carrier protein synthase of Pseudomonas aeruginosa, an organism that harbors merely one PPTase, namely PcpS. Gene deletion experiments clearly show that PcpS is essential for growth of P. aeruginosa, and biochemical data indicate its association with both fatty acid synthesis and siderophore metabolism. At first sight, PcpS is a PPTase of the monomeric Sfp-type and was consequently expected to have catalytic properties typical for this type of enzyme. However, in vitro characterization of PcpS with natural protein partners and non-cognate substrates revealed that its catalytic properties differ significantly from those of Sfp. Thus, the situation in P. aeruginosa is not simply the result of the loss of an AcpS-type PPTase. PcpS exhibits high catalytic efficiency with the carrier protein of fatty acid synthesis and shows a reduced although significant conversion rate of the carrier proteins of non-ribosomal peptide synthetases from their apo to holo form. This association with enzymes of primary and secondary metabolism indicates that PcpS belongs to a new sub-class of PPTases.     

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。     

Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 microm and slightly inhibited at concentrations of 5-10 microm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3',5'-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4'-phosphopantetheine to the apo-ACP of the complex.     

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation‐PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C‐terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. Proteins 2014; 82:2691–2702. © 2014 Wiley Periodicals, Inc.     

A genome rearrangement has orphaned the Escherichia coli K-12 AcpT phosphopantetheinyl transferase from its cognate Escherichia coli O157:H7 substrates

Phosphopantetheinyl transferases (PPTases) are enzymes that catalyse the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of a carrier protein. These carrier proteins use the 4'-phosphopantetheine thiol to shuttle intermediates between the active sites of biosynthetic enzymes involved in fatty acid, non-ribosomal peptide and polyketide synthesis. Three PPTases have been previously been identified in Escherichia coli K-12 and other E. coli strains by homology searches and are encoded by the genes acpS, entD and acpT. Both AcpS and EntD have been well studied whereas the function of AcpT has been an enigma because no carrier protein substrate could be found. We report genetic and biochemical evidence that AcpT modifies two carrier proteins encoded in O-island 138, a cluster of fatty acid biosynthesis-like genes located adjacent to acpT in the genome of the pathogenic E. coli strain O157:H7 (E. coli K-12 and several other sequenced E. coli and Shigella strains lack O-island 138). The two carrier proteins of O-island 138 of strain O157:H7 are not modified (or only very poorly modified) by AcpS, the PPTase responsible for 4'-phosphopantetheine attachment to the acyl carrier protein (AcpP) of fatty acid synthesis. We demonstrate that AcpT cannot functionally replace AcpS in E. coli K-12 either in its native chromosomal location or upon insertion of acpT into the acpS chromosomal location. However, in the absence of AcpS activity AcpT does allow very slow growth thus providing a rationale for its retention in the absence of its cognate substrates. These results together with phylogenetic analyses and comparisons of the E. coli and Shigella strains of known genome sequence strongly argue that AcpT has been orphaned from its cognate substrates by a deletion event that occurred in a common ancestor of these organisms. This seems one of the few cases where a chromosomal rearrangement has been functionally demonstrated to be a deletion event rather than an insertion event in the reference organism. We also show that the previously reported suppression of an acpS mutation by the deletion of Lon protease is an artifact of the increased capsular polysaccharide production of lon strains.     

The phosphopantetheinyl transferase KirP activates the ACP and PCP domains of the kirromycin NRPS/PKS of Streptomyces collinus Tü 365

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis.     

Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes responsible for the synthesis of a variety of microbial bioactive peptides. They consist of modules that each recognise and incorporate one specific amino acid into the peptide product. A module comprises several domains, which carry out the individual reaction steps. After activation by the adenylation domain, the amino acid substrate is covalently tethered to a 4'-phosphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes the substrate to the reaction centres of the consecutive domains. RESULTS: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techniques. PCP is a distorted four-helix bundle with an extended loop between the first two helices. Its overall fold resembles the topology of acyl carrier proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodin polyketide synthase from Streptomyces coelicolor; however, the surface polarity and the length and relative alignment of the helices are different. The conserved serine, which is the cofactor-binding site, has the same location as in the ACPs and is situated within a stretch of seven flexible residues. CONCLUSIONS: The structure of PCP reflects its character as a protein domain. The fold is well defined between residues 8 and 82 and the structural core of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine. The flexibility of the post-translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains.     

The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins

Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl‐carrier proteins (PCPs) or acyl‐carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs. Proteins 2014; 82:1210–1218. © 2013 Wiley Periodicals, Inc.     

Portability of epimerization domain and role of peptidyl carrier protein on epimerization activity in nonribosomal peptide synthetases.

Incorporation of nonproteinogenic amino acids in small polypeptides synthesized by nonribosomal peptide synthetases (NRPS) significantly contributes to their biological activity. In these peptides, conversion of L-amino acids to the corresponding D-isomer is catalyzed by specialized NRPS modules that utilize an epimerization (E) domain. To understand the basis for the specific interaction of E domains with PCP domains (peptidyl carrier proteins, also described as T domains) and to investigate their substrate tolerance, we constructed a set of eight fusion proteins. The gene fragments encoding E and PCP-E domains of TycA (A-PCP-E), the one module tyrocidine synthetase A, were fused to different gene fragments encoding A and A-PCP domains, resulting in A/PCP-E and A-PCP/E types of fusion proteins (slash indicates site of fusion). We were able to show that the E domain of TycA, usually epimerizing Phe, does also accept the alternate substrates Trp, Ile, and Val, although with reduced efficiency. Interestingly, however, an epimerization activity was only observed in the case of fusion proteins where the PCP domain originates from modules containing an E domain. Sequence comparison revealed that such PCPs possess significant differences in the signature Ppant binding motif (CoreT: [GGDSI]), when compared to those carrier proteins, originating from ordinary C-A-PCP elongation modules (CoreT: [GGHSL]). By means of mutational analysis, we could show that epimerization activity is influenced by the nature of amino acid residues in proximity to the cofactor Ppant binding site. The aspartate residue in front of the invariant serine (Ppant binding site) especially seems to play an important role for the proper interaction between PCP and the E domain, as well as the presentation of the aminoacyl-S-Ppant substrate in the course of substrate epimerization. In conclusion, specialized PCP domains are needed for a productive interaction with E domains when constructing hybrid enzymes.     

Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis

Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3'5'-ADP, a product of AcpS, at 2.0 and 1.9 A resolution, respectively. The crystal structure reveals an alpha/beta fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity.     

Functional characterization of an evolutionarily distinct phosphopantetheinyl transferase in the apicomplexan Cryptosporidium parvum

Recently, two types of fatty acid synthases (FASs) have been discovered from apicomplexan parasites. Although significant progress has been made in characterizing these apicomplexan FASs, virtually nothing was previously known about the activation and regulation of these enzymes. In this study, we report the discovery and characterization of two distinct types of phosphopantetheinyl transferase (PPTase) that are responsible for synthesizing holo-acyl carrier protein (ACP) from three apicomplexan parasites: surfactin production element (SFP) type in Cryptosporidium parvum (CpSFP-PPT), holo-ACP synthase (ACPS)-type in Plasmodium falciparum (PfACPS-PPT), and both SFP and ACPS types in Toxoplasma gondii (TgSFP-PPT and TgACPS-PPT). CpSFP-PPT and TgSFP-PPT are monofunctional, cytosolic, and phylogenetically related to animal PPTases. However, PfACPS-PPT and TgACPS-PPT are bifunctional (fused with a metal-dependent hydrolase), likely targeted to the apicoplast, and more closely related to proteobacterial PPTases. The function of apicomplexan PPTases has been confirmed by detailed functional analysis using recombinant CpSFP-PPT expressed from an artificially synthesized gene with codon usage optimized for Escherichia coli. The recombinant CpSFP-PPT was able to activate the ACP domains from the C. parvum type I FAS in vitro using either CoA or acetyl-CoA as a substrate, or in vivo when coexpressed in bacteria, with kinetic characteristics typical of PPTases. These observations suggest that the two types of fatty acid synthases in the Apicomplexa are activated and regulated by two evolutionarily distinct PPTases.     

Chain initiation in the leinamycin-producing hybrid nonribosomal peptide/polyketide synthetase from Streptomyces atroolivaceus S-140. Discrete, monofunctional adenylation enzyme and peptidyl carrier protein that directly load D-alanine

Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiation module, an adenylation (A) domain activates an amino acid and installs it onto a peptidyl carrier protein (PCP) domain as a thioester; an elongation module, which has a condensation (C) domain located between every consecutive pair of A and PCP domains, catalyzes the formation of the peptide bond between the upstream aminoacyl/peptidyl-S-PCP and the free amino group of the downstream aminoacyl-S-PCP. D-amino acid constituents in peptide natural products usually arise from the L-enantiomers through the action of integral epimerization (E) domains of an NRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a D-alanine moiety, does not encode a typical NRPS initiation module with the expected A-PCP-E domains; instead, it has only an A protein (LnmQ) and a PCP (LnmP), both of which are encoded by separate genes. Here we show the results of biochemical experiments as follows: (i) we demonstrate that LnmQ directly activates D-alanine as D-alaninyl-AMP and installs it onto LnmP to generate a D-alaninyl-S-PCP intermediate; (ii) we confirm that aminoacylation of LnmP by LnmQ in trans is the result of specific communication between the separate A and PCP proteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous D-alanine in the fermentation broth of Streptomyces atroolivaceous S-140. These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete D-alanine-specific A protein and a PCP.     

Two functionally distinctive phosphopantetheinyl transferases from amoeba Dictyostelium discoideum

The life cycle of Dictyostelium discoideum is proposed to be regulated by expression of small metabolites. Genome sequencing studies have revealed a remarkable array of genes homologous to polyketide synthases (PKSs) that are known to synthesize secondary metabolites in bacteria and fungi. A crucial step in functional activation of PKSs involves their post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases). PPTases have been recently characterized from several bacteria; however, their relevance in complex life cycle of protozoa remains largely unexplored. Here we have identified and characterized two phosphopantetheinyl transferases from D. discoideum that exhibit distinct functional specificity. DiAcpS specifically modifies a stand-alone acyl carrier protein (ACP) that possesses a mitochondrial import signal. DiSfp in contrast is specific to Type I multifunctional PKS/fatty acid synthase proteins and cannot modify the stand-alone ACP. The mRNA of two PPTases can be detected during the vegetative as well as starvation-induced developmental pathway and the disruption of either of these genes results in non-viable amoebae. Our studies show that both PPTases play an important role in Dictyostelium biology and provide insight into the importance of PPTases in lower eukaryotes.