Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin.

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein-protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein-protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein-protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and ß-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min-1 mg-1. FabD R117A maintained K M values for holo-ACP (~ 40 µM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 µM) and acetyl-CoA (200 ± 70 µM) when compared to malonyl-CoA (80 ± 30 µM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Improvement of FK506 production in Streptomyces tsukubaensis by genetic enhancement of the supply of unusual polyketide extender units via utilization of two distinct site-specific recombination systems.

FK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain Streptomyces tsukubaensis by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that S. tsukubaensis is amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.

[Overexpression and purification of Escherichia coli holo-acyl carrier protein and synthesis of acyl carrier protein].

OBJECTIVE: To investigate the mechanism of fatty acids, lipid A and N-acylhomoserine lactones biosynthesis of bacteria by using high quality Escherichia coli holo-ACP and varied length chain acyl-ACPs as substrates. METHODS AND RESULTS: Using PCR technique we amplified the acpP and acpS gene fragments from genomic DNA of E. coli strain MG1655. Ligating these gene fragments with plasmids pBAD24 or pET28b respectively, we obtained 3 expression plasmids of acyl carrier protein: pBAD-ACP, pET-ACP and pET-ACP-ACPS, and one expression plasmid of holo-acyl carrier protein synthase: pBAD-ACPS. Then we constructed 3 acyl carrier protein producer strains: DH5alpha/pBAD-ACP, BL21 (DE3)/pET-ACP and BL21(DE3)/pET-ACP-ACPS by transforming E. coli strains DH5alpha or BL21(DE3)with pBAD-ACP, pET-ACP or pET-ACP-ACPS, respectively. Although these 3 strains could produce more acyl carrier protein under induction than strain DK574, which was used to purify holo-acyl carrier protein in general, the yield of holo-acyl carrier protein of these strains was still lower. In order to increase the yield of holo-acyl carrier protein in these strains, we introduced pBAD-ACPS into these strains. The assay of expressions of new strains was shown the that strain DH5alpha harbored pBAD-ACP and pBAD-ACPS double plasmids produced more holo-acyl carrier protein than strain DK574, and the purity of holo-acyl carrier protein was also increased (up to 99%). Then we purified high quality holo-acyl carrier protein from the culture of the strain DH5alpha harbored pBAD-ACP and pBAD-ACPS by using UNOsphere Q anion-exchange chromatography. Utilizing holo-acyl carrier protein and long chain fatty acids as substrates and under Vibrio harveyi acyl-acyl carrier protein synthetase catalyzing, we synthesized several different acyl-acyl carrier proteins. CONCLUSION: From this study we obtained a high holo-ACP producer strain and demonstrated that co-expressing acpP with acpS, E. coli strains could produce more holo-ACP.

Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.

BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.

Biochemical characterization of a malonyl-specific acyltransferase domain of FK506 biosynthetic polyketide synthase.

Acyltransferases (ATs) play an essential role in the polyketide biosynthesis through transferring acyl units into acyl carrier proteins (ACPs) via a self-acylation reaction and a transacylation reaction. Here we used AT10FkbA of FK506 biosynthetic polyketide synthase (PKS) from Streptomyces tsukubaensis YN06 as a model to study the specificity of ATs for acyl units. Our results show that AT10FkbA can form both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction, however, only malonyl-O-AT10FkbA but not methylmalonyl-O-AT10FkbA can transfer the acyl unit into ACPs in the transacylation reaction. Unlike some ATs that are known to control the acyl specificity in self-acylation reactions, AT10FkbA controls the acyl specificity in transacylation reactions.

Overproduction of the acyl carrier protein component of a type II polyketide synthase stimulates production of tetracenomycin biosynthetic intermediates in Streptomyces glaucescens.

The development of microorganisms with improved antibiotic production is an important goal in the commercialization of new pharmaceuticals or in lowering the cost of established drugs. We report a way to achieve this for biosynthetic intermediates of an antibiotic made by the polyketide pathway whose earliest steps involve a Type II multienzyme complex. Introduction of the tcmKLM beta-ketoacyl: ACP synthase and acyl carrier protein (ACP) genes or just the tcmM ACP gene into the tetracenomycin (Tcm) C-producing Streptomyces glaucescens wild-type strain, or its tcmN or tcmO blocked mutants, on high copy vectors under the control of strong promoters caused a 2 to 30-fold overproduction of Tcm D3 and some other biosynthetic intermediates (or shunt products) and a 25 to 30% increase in Tcm C production relative to the control strains carrying the plasmid vector only. However, Tcm C production was not greater than that obtained with the vector-free wild-type strain. The unexpected effect of increased ACP on Tcm D3 production suggests that the level of this protein can influence either the activity or level of the three other components of the Tcm polyketide synthase.

To cyclize or not to cyclize: catching enzyme evolution in the act.

If you look at the biggest genes in soil and marine bacteria, you tend to see the chemical blueprints for making natural products such as peptides and polyketides. Over the past decade, collective efforts of enzymologists working with synthetic and analytical chemists have been catching up with the data dump from microbial genome sequencing. Following this story line, we now understand how cyanobacteria construct scaffolds for the related natural products curacin and jamaicamide using subtle tweaks to non-standard biosynthetic machinery.

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning the oxazolomycin biosynthetic gene cluster from Streptomyces albus JA3453.

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A.

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.

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.

A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum.

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton-nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods.

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene.

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.

Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases.

6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB, the aglycon core of the antibiotic erythromycin. The biosynthesis of 6-dEB proceeds in an assembly-line fashion through the six modules of DEBS, each of which catalyzes a dedicated set of reactions, such that the structure of the final product is determined by the arrangement of modules along the assembly line. This transparent relationship between protein sequence and enzyme function is common to all modular PKSs and makes these enzymes an attractive scaffold for protein engineering through module swapping. One of the fundamental issues relating to module swapping that still needs to be addressed is the mechanism by which intermediates are channeled from one module to the next. While it has been previously shown that short linker regions at the N- and C-termini of adjacent polypeptides play an important role in mediating intermodular transfer, the contributions of other protein-protein interactions have not yet been probed. Here, we investigate the roles of the linker interactions as well as the interactions between the donor acyl carrier protein (ACP) domain and the downstream ketosynthase (KS) domain in various contexts. Linker interactions and ACP-KS interactions make relatively equal contributions at the module 2-module 3 and the module 4-module 5 interfaces in DEBS. In contrast, modules 2 and 6 are more tolerant toward substrates presented by nonnatural ACP domains. This tolerance was exploited for engineering hybrid PKS-PKS and PKS-NRPS (nonribosomal peptide synthetase) junctions and suggests fundamental ground rules for engineering novel chimeric PKSs in the future.

Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B.

A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus. The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit. Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.

Acyl carrier protein (ACP) import into chloroplasts. Covalent modification by a stromal holoACP synthase is stimulated by exogenously added CoA and inhibited by adenosine 3',5'-bisphosphate.

During the import of the precursor for the acyl carrier protein (ACP) into chloroplasts, apoACP is converted to holoACP by the attachment of a phosphopantetheine group transferred from coenzyme A (CoA) by a chloroplast holoACP synthase [Fernandez, M. and Lamppa, G. (1990) Acyl carrier protein import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holoACP synthase, Plant Cell 2, 195-206]. Here it is shown that exogenous addition of CoA to intact chloroplasts in the import assay stimulates the conversion of apoACP to holoACP. If adenosine 3',5'-bisphosphate [Ado(3',5')P2], the byproduct of the transfer reaction, was also included the extent of conversion was greatly reduced. CoA has its effect after ACP precursor (pre-ACP) import and proteolytic removal of the transit peptide, thus indicating that the chloroplast holoACP synthase resides in the stroma where fatty acid synthase is found. When Ado(3',5')P2 was added alone to the import assay, it inhibited the synthesis of holoACP. Inhibition of the conversion of apo- to holoACP with Ado(3',5')P2 made it possible to examine whether the holoform of preACP could be imported into chloroplasts. Pre-apoACP was synthesized in Escherichia coli and shown to be competent for import in an ATP- and temperature-dependent manner. A partially purified chloroplast holoACP synthase converted 60-90% of the pre-apoACP to pre-holoACP. Pre-holoACP incubated with chloroplasts in the presence of Ado(3',5')P2 yielded > 60% holoACP, whereas the control reaction with pre-apoACP gave primarily apoACP. Hence the phosphopantetheine prosthetic group of ACP does not block precursor movement through the translocation apparatus of the chloroplast envelope.

Plastid-localised seed acyl-carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family.

Acyl-carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N-terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 51-amino-acid N-terminal extension. Immunocytochemical studies showed ACP to be localised solely within the plastids of B. napus seed tissue and it would therefore appear that the N-terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.

Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro.

Avena seedlings grown in the presence of the plant tetrapyrrole synthesis inhibitor 4-amino-5-hexynoic acid contain less than 10% of the spectrally detectable phytochrome levels found in untreated seedlings, but continue to accumulate phytochrome apoprotein (Elich, T. D., and Lagarias, J. C. (1988) Plant Physiol. 88, 747-751). Using such tetrapyrrole-deficient seedlings, we have previously reported that phycocyanobilin, the cleaved prosthetic group of C-phycocyanin, can be incorporated into phytochrome in vivo to yield spectrally active holoprotein (Elich, T. D., McDonagh, A. F., Palma, L. A., and Lagarias, J. C. (1988) J. Biol. Chem. 264, 183-189). Here we show that addition of phycocyanobilin to soluble extracts of inhibitor-treated seedlings results in a rapid increase in spectrally active phytochrome holoprotein. The newly formed photoactive species displays a blue-shifted absorbance difference spectrum similar to that observed in the previous in vivo studies. The increase in spectral activity is consistent with conversion of all of the preexisting phytochrome apoprotein to functionally active holoprotein. The formation of a covalent phycocyanobilin-apophytochrome adduct is shown by an increase in Zn2+-dependent bilin fluorescence of the phytochrome polypeptide. A photoreversible, covalent adduct with a similar optical spectrum also forms when immunopurified apophytochrome is incubated with phycocyanobilin. ATP, reduced pyridine nucleotides, or other cofactors are not required for adduct formation. When biliverdin IX alpha is substituted for phycocyanobilin, no spectrally active covalent adduct is produced. These results indicate that an A-ring ethylidene-containing bilatriene is required for post-translational covalent attachment of bilin to apophytochrome and that apophytochrome may be the bilin C-S lyase which catalyzes bilin attachment.