The carrier protein domain of BpsA adopts a characteristic fold, often consisting of a four-helix bundle or a similar structural motif. This fold presents the conserved serine residue in a specific orientation, facilitating its recognition by PPTases.
Surrounding the conserved serine residue, there are often hydrophobic and charged surface residues that contribute to the binding and recognition of PPTases. These residues can be targeted for mutagenesis during directed evolution to enhance the interaction with non-cognate PPTases. Some carrier protein domains contain flexible linker regions or loops that may play a role in the binding and positioning of the carrier protein within the PPTase active site. These regions can also be targeted for mutagenesis. During activation by PPTases the transfer of the 4'-phosphopantetheinyl moiety from coenzyme A (CoA) to the conserved serine residue on the carrier protein domain takes place and this chemical transformation is catalyzed by PPTases and proceeds through a series of steps. Let us analyse this process deeper and eventually, explore a couple entirely new possibilities.
First the Binding of CoA takes place. The PPTase binds to CoA, positioning the 4'-phosphopantetheinyl group for transfer. This binding is facilitated by specific interactions between the enzyme and the CoA molecule. The PPTase recognizes and binds to the apo-form of the carrier protein, specifically targeting the conserved serine residue. This recognition is mediated by the structural features of the carrier protein domain and the complementary binding site on the PPTase. The PPTase catalyzes the nucleophilic attack of the serine hydroxyl group on the phosphopantetheine moiety of CoA, resulting in the formation of a new phosphoester bond. This reaction is facilitated by the precise positioning of the reactants within the enzyme's active site. Since modified carrier protein, now in its active holo-form, is released from the PPTase, ready to participate in the biosynthetic pathway, exploring these proteins further, we note the specificity of PPTases for their cognate carrier proteins is determined by a combination of structural and chemical factors. Understanding these determinants is crucial for engineering BpsA to be recognized by non-cognate PPTases. Let us dive deeper into these molecular actions. The active site of PPTases is shaped to accommodate the specific structural features of their cognate carrier proteins. Mutations that alter the active site geometry can at times expand the substrate specificity of PPTases. The surface residues of both the PPTase and the carrier protein contribute to the specificity of their interaction. Mutations that modify the surface properties, such as charge distribution or hydrophobicity, can influence the binding affinity and specificity.
Hydrogen bonding interactions between the PPTase and the carrier protein play a crucial role in substrate recognition and binding. Mutations that alter these hydrogen bonding networks can modulate the specificity of the interaction. Charged residues on the PPTase and the carrier protein can form electrostatic interactions that contribute to the specificity of the interaction. We have the option to modulate these charge distributions through mutagenesis and we can hence likely expand the substrate specificity of PPTases. Hydrophobic interactions between the PPTase and the carrier protein can also influence the specificity of the interaction. Mutations that alter the hydrophobic surfaces of the interacting partners can sometimes enhance or disrupt these interactions. One of the primary outcomes of directed evolution is the expansion of the substrate scope for BpsA. By engineering BpsA to be recognized and activated by a broader range of PPTases, the enzyme can incorporate a wider variety of building blocks into the growing polyketide or non-ribosomal peptide chain. The ability to utilize non-cognate PPTases can enable BpsA to interact with polyketide synthases (PKSs) that it would not normally recognize. This can lead to the production of polyketides with new kind of structural features, such as modified carbon skeletons, altered stereochemistry, or the incorporation of non-canonical building blocks. Similarly, the evolved BpsA variants may be able to participate in the biosynthesis of non-ribosomal peptides (NRPs) catalyzed by non-cognate non-ribosomal peptide synthetases (NRPSs). This could result in the synthesis of peptides with novel amino acid compositions, modified side chains, or new cyclization patterns.
In some cases, the expanded substrate scope of BpsA may enable the production of hybrid molecules that combine features of both polyketides and non-ribosomal peptides. These hybrid structures can exhibit new types of biological activities and interact with new therapeutic targets. The introduction of evolved BpsA variants into microbial hosts can lead to the activation of previously silent or cryptic biosynthetic pathways. Those pathways may have been inactive due to the lack of appropriate PPTase recognition, but the engineered BpsA can now facilitate their expression. Microbial genomes should not be overlooked since it is known many microbial genomes contain cryptic gene clusters encoding biosynthetic pathways for potent and valuable natural products. By providing the necessary activation through evolved BpsA, these cryptic pathways can be unlocked. The evolved BpsA variants may enable the mixing and matching of enzymes from different biosynthetic pathways, resulting in the combinatorial biosynthesis of new molecules. This can lead to the generation of hybrid structures with one of a kind chemical scaffolds and perhaps enhanced biological activities. The evolved BpsA can also influence the stereochemistry and regioselectivity of biosynthetic reactions. Subtle changes in the positioning of intermediates or the orientation of active sites can lead to the formation of new stereoisomers or the preferential incorporation of building blocks at different positions. Considering these natural products, Polyketides are a good example, since they are a diverse class of natural products with a wide range of biological activities, including antibacterial, antifungal, and anticancer properties. The evolved BpsA variants can usually interact with polyketide synthases (PKSs) that were previously incompatible with the native enzyme, enabling the synthesis of polyketides with novel structural features. It is also possible for the evolved BpsA to facilitate the incorporation of non-canonical building blocks into the growing polyketide chain. This can result in modified carbon skeletons and unique structural scaffolds, which may exhibit new biological activities. As mentioned before, the actual mutations might influence the stereochemistry and this can lead to the production of polyketides with different stereoisomeric configurations, usually enhancing their biological efficacy or specificity. The enhanced activation of BpsA by non-cognate PPTases may enable interactions with tailoring enzymes, such as oxidoreductases, methyltransferases, or glycosyltransferases and in turn these interactions can introduce additional chemical modifications to the polyketide structures, further diversifying their chemical and biological properties.
The evolved BpsA variants can potentially interact with non-ribosomal peptide synthetases (NRPSs) that were previously incompatible, leading to the synthesis of novel NRPs and the evolved BpsA may enable the incorporation of non-proteinogenic amino acids or modified amino acid building blocks into the growing peptide chain. This can result in NRPs with a whole new set of structural features and novel biological activities. Interactions between the evolved BpsA and NRPSs could influence the cyclization and macrocyclization patterns of the synthesized peptides, leading to the formation of novel cyclic and macrocyclic structures, which are often associated with enhanced stability and bioactivity. The production of hybrid molecules that combine features of both polyketides and non-ribosomal peptides is likely. These hybrid structures can exhibit one of a kind biological activities and interact with new therapeutic targets. NRPs, are in fact a class of secondary metabolites with diverse biological activities, (antibiotics, immunosuppressants, anticancer agents...) and are currently under investigation.
Novel compounds
Specialized metabolites, such as siderophores and signalling molecules, play crucial roles in microbial physiology and survival. The evolved BpsA variants can interact with biosynthetic enzymes involved in the production of these specialized metabolites, leading to the discovery of novel compounds.
The evolved BpsA may enable the synthesis of siderophores with modified iron-chelating moieties or altered structural features. Those novel siderophores could exhibit enhanced iron-binding properties or improved bioavailability, making them precious for therapeutic or agricultural applications. Interactions between the evolved BpsA and enzymes involved in quorum sensing pathways could lead to the production of novel signalling molecules and while said molecules could modulate microbial communication and behavior, for now the question remains open, if these molecules could offer new strategies for controlling bacterial infections or biofilm formation. The exploration of specialized metabolite biosynthesis using the evolved BpsA variants may uncover some novel antimicrobial compounds with entirely new modes of action, and because these compounds could contribute to the development of new antibiotics, their analysis should have priority since in this way we could address the growing challenge of antibiotic resistance.
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