Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing.
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Amino Acid Sequence↗
Biomedical subjects
Henning D Mootz
Publications and source records attributed to Henning D Mootz.
Engineering artificially split inteins for applications in protein chemistry: biochemical characterization of the split Ssp DnaB intein and comparison to the split Sce VMA intein.
In protein trans-splicing, an intein domain split into two polypeptide chains mediates linkage of the flanking amino acid sequences, the N- and C-terminal exteins, with a native peptide bond. This process can be exploited to assemble proteins from two separately prepared fragments, e.g., for the segmental labeling with isotopes for NMR studies or the incorporation of chemical and biophysical probes. Split inteins can be artificially generated by genetic means; however, the purified inteinN and inteinC fragments usually require a denaturation and renaturation treatment to fold into the active intein, thus preventing their application to proteins that cannot be refolded. Here, we report that the purified fragments of the artificially split DnaB helicase of Synechocystis spp. PCC6803 (Ssp DnaB) intein are active under native conditions. The first-order rate constant of the protein trans-splicing reaction was 7.1 x 10(-4) s(-1). The previously described split vacuolar ATPase of Saccharomyces cerevisiae (Sce VMA) intein is the only other artificially split intein that is active under native conditions; however, it requires induced complex formation of the intein fragments by auxiliary dimerization domains for efficient protein trans-splicing. In contrast, fusion of the dimerization domains to the split Ssp DnaB intein fragments had no effect on activity. This difference was also reflected by a higher thermostability of the split Ssp DnaB intein. Further investigations of the split Sce VMA intein under optimized conditions revealed a first-order rate constant of 9.4 x 10(-4) s(-1) for protein trans-splicing and 1.7 x 10(-3) s(-1) for C-terminal cleavage involving a Cys1Ala mutant. Finally, we show that the two split inteins are orthogonal, suggesting further applications for the assembly of proteins from more than two parts.
Adenosine Triphosphatases↗
Probing the molecular basis of protein function through chemistry.
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Molecular Probes↗
Design of an intein that can be inhibited with a small molecule ligand.
Protein splicing is a process in which an intervening sequence, the intein, catalyzes its own excision out of a larger polypeptide precursor by joining the flanking sequences, the exteins, with a native peptide bond. Inteins are almost completely promiscuous toward the nature of their extein sequences and can be inserted into virtually any host protein. The intein-mediated formation of a peptide bond between two polypeptides offers great potential to modulate protein structure and, hence, protein function on the post-translational level. In this work, we report the design of an intein that can be inhibited by the addition of a specific small molecule ligand. Our design strategy involved the generation of a trans-splicing intein, in which the intein domain is split into two-halves that are located on two separate polypeptides, each joined with the respective N- or C-terminal extein. To turn these fragments into an active intein with an incorporated "off" switch, each was fused at its newly created terminus with the F36M mutant of FKBP12, referred to as the FM domain. The F36M substitution was reported to effect a homodimerization of the usually monomeric FKBP12 protein; however, addition of the small molecule ligand, rapamycin, or synthetic derivatives thereof leads to a dissociation of the dimer. This phenomenon was exploited by first reconstituting the active intein on the basis of FM domain dimerization. Second, addition of the small molecule ligand prevented formation of the active intein complex and inhibited protein trans-splicing. This intein exhibited unexpected kinetic properties and provides a new and potentially very general means to control protein function on the post-translational level.
Carrier Proteins↗
Activation of an autoregulated protein kinase by conditional protein splicing.
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Amino Acid Sequence↗
In vivo production of artificial nonribosomal peptide products in the heterologous host Escherichia coli.
Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4'-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.
Anti-Bacterial Agents↗
Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo.
Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.
Animals↗
Systematic and quantitative analysis of protein-protein recognition between nonribosomal peptide synthetases investigated in the tyrocidine biosynthetic template.
We present a systematic and quantitative study of the protein-protein recognition between the three tyrocidine synthetases TycA, TycB, and TycC investigated with two artificial in trans assay systems, which had been previously developed: the "DKP assay system" for the interaction of TycA with TycB and the "L/D-Phe-L-Asn assay system" for the interaction of TycB with TycC. TycA-A(Phe)TE and TycB(3)-A(Phe)TE, which are used as donor enzymes, both provide D-Phe-S-Ppant, so that no substrate specificities interfered with the quantification of protein-protein recognition. We tested all donor/acceptor enzyme combinations between the two artificial assay systems for product formation activities as well as two hybrid enzymes, where the E-domains of TycA and TycB(3) had been exchanged against each other. Furthermore, four donor/acceptor protein fusions were constructed on gene level, resulting in dimodular proteins. We were able to show that the E-domains mediate protein-protein recognition in trans. Product formation of the different donor assayed with the two acceptor enzymes TycB(1)-CA(Pro)T and TycC(1)-CA(Asn)T/Te in trans was only obtained if the donor enzyme harbored the cognate E-domain. Interestingly, all in cis fusions (dimodular proteins) were active, giving strong evidence that unnatural protein-protein interactions can be "forced" by fusion of the distinct enzymes. Finally, we were able to detect product formation in the "DKP system" with engineered hybrid proteins where the A-domain of TycA had been exchanged against the isoleucine-activating A-domain of BacA(1) and the valine-activating A-domain of TycC(4), respectively. All of these findings are of high relevance for future NRPS engineering approaches.
Anti-Bacterial Agents↗
Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases.
Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf). These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.
Acyl Carrier Protein↗
Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes.
Many natural products of therapeutical and biotechnological importance are nonribosomally synthesized peptides. Structural hallmarks of this class of compounds are the occurrence of unusual amino acids, mostly cyclic peptide backbones, and numerous further modifications such as acylation, heterocyclic ring formation, and glycosylation. Because of their structural complexity, chemical synthesis is usually an unattractive route to these molecules. In contrast, genetic engineering of the biosynthesis genes emerges as a potentially powerful approach to the combinatorial biosynthesis of useful analogues of the lead compounds. Nonribosomal peptide synthetases (NRPSs) carry out a sequential multistep assembly and modification of the peptides in a thiotemplate process described by the multiple carrier model. The modular architecture of NRPSs suggests straightforward methods for the reprogramming of these enzymes by exchange of catalytic subunits. However, many of the reported engineering attempts faced low product yields or even inactive hybrid enzymes. Using a new approach to obtain hybrid NRPSs, we show here that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. Furthermore, a module exchange resulted in a significantly higher product yield than that observed in previous studies.
Anti-Bacterial Agents↗
Protein splicing triggered by a small molecule.
The use of small molecules that turn specific proteins on or off provides a level of temporal control that is difficult to achieve using standard genetic approaches. Consequently, the development of small-molecule switches of protein function is a very active area of chemical biology, sometimes referred to as chemical genetics. Most studies in this area rely on the identification of small molecules that bind directly to the active site of a target protein, thereby acting as agonists or antagonists of function. Strategies have also been described in which the small molecule triggers a change in the secondary, tertiary, or ternary structure of the protein, in so doing changing the functional state of the molecule. Another approach to this problem would be to alter the primary structure of a target protein in response to a small-molecule trigger; a dramatic change in primary sequence would be directly coupled to function. In principle, this can be achieved by harnessing protein splicing, a posttranslational editing process that results in the precise removal of an internal domain (termed an intein) from two flanking sequences termed the N- and C-exteins. In this communication we introduce a technique that allows protein splicing to occur only in the presence of the small molecule, rapamycin. This approach is expected to be independent of the nature of the two exteins and so should provide a general vehicle for controlling protein function using small molecules.
Blotting, Western↗
Functional characterization of 4'-phosphopantetheinyl transferase genes of bacterial and fungal origin by complementation of Saccharomyces cerevisiae lys5.
Lysine biosynthesis in yeast requires the posttranslational conversion of the alpha-aminoadipate semialdehyde reductase Lys2 by the 4'-phosphopantetheinyl transferase (PPTase) Lys5 from the inactive apo-form into the catalytically active holo-form. In this reaction, the peptidyl carrier domain of Lys2 is modified at a conserved serine residue side chain with the 4'-phosphopantetheine moiety derived from coenzyme A. We have deleted the lys5 gene in Saccharomyces cerevisiae to investigate the substrate specificity of various heterologous PPTase genes of bacterial and fungal origin by testing their ability to complement lys5 in trans. Genes encoding PPTases Sfp and Gsp from Bacillus spp., which are involved in non-ribosomal peptide antibiotic synthesis, complemented the lys5 deletion, whereas ydcB of Bacillus subtilis, which encodes the acyl carrier protein synthase involved in fatty acid synthesis, could not. Two yet uncharacterized fungal genes, q10474 of Schizosaccharomyces pombe, meanwhile annotated as the putative lys7 gene, and npgA of Aspergillus nidulans, also complemented the lys5 deletion and have thus been functionally characterized as PPTases. The complementation system described also provides the basis for a simple method of functional characterization of PPTase candidate genes and their cloning from chromosomal DNA or cDNA libraries of diverse origin.
Aspergillus↗
Ways of assembling complex natural products on modular nonribosomal peptide synthetases.
Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a large number of complex peptide natural products, many of which display therapeutically useful activity. Each cycle of chain extension is carried out by a dedicated module of the multifunctional enzymes. A module harbors all the catalytic units, which are referred to as domains, necessary for recognition, activation, covalent binding, and optionally modification of a single building block monomer, as well as for peptide-bond formation with the growing chain. A terminal domain releases the full-length peptide chain from the enzyme complex. Recent characterization of many NRPS systems revealed several examples where the sequence of the product does not directly correspond to the linear arrangement of modules and domains within the enzyme(s). It is now obvious that these systems cannot be regarded as rare exceptions of the common NRPS architecture but rather represent more complicated variations of the NRPS repertoire to increase their biosynthetic potential. In most of these cases unusual peptide structures of the products are observed, such as structures with side-chain acylation, cyclization involving the peptide backbone and/or side chains, and transfer of the peptide chain onto soluble small-molecule substrates. These findings indicate a previously unexpected higher versatility of the modules and domains in terms of both catalytic potential and interaction within the multifunctional protein templates. We propose to classify the known NRPS systems into three groups, linear NRPSs (type A), iterative NRPSs (type B), and nonlinear NRPSs (type C), according to their biosynthetic logic. Understanding the various biosynthetic strategies of NRPSs will be crucial to fully explore their potential for engineered combinatorial biosynthesis.
Acylation↗