The BpsA carrier protein domain is integral to the biosynthesis of polyketides, a class of secondary metabolites with diverse biological activities. The carrier protein domain facilitates the post-translational modification of peptide substrates, which is essential for their biological activity. The activation of BpsA involves the attachment of a 4'-phosphopantetheine group to a serine residue, a modification catalyzed by PPTases.
In order to activate carrier proteins we need to analyse PPTases. PPTases are the enzymes that catalyze the transfer of the 4'-phosphopantetheine moiety from coenzyme A to specific serine residues on carrier proteins like BpsA. This modification is critical as it converts the inactive apo-form of the carrier protein to the active holo-form, enabling the carrier protein to participate in biosynthetic pathways. Non-cognate PPTases, which are not the natural partners of BpsA, offer the potential to expand the range of biochemical pathways that BpsA can participate in, hereby increasing the diversity of bioactive compounds that can be synthesized.
We want to engineer proteins (mimicking the process of natural selection), so we need to generate a library of protein variants through mutagenesis and screening these variants for desired traits we can move onward, iterating this process to enhance particular properties of the protein in question, in our case to develop new medicine for the ASD patient.
We can employ several key methods to develop such a drug. One such method is Error-Prone PCR, which introduces random mutations into the BpsA gene to create a diverse library of variants. Through DNA Shuffling, we can recombine fragments of BpsA gene variants to create new combinations of mutations. Additionally, CRISPR-Based Mutagenesis utilizes the CRISPR-Cas9 system to introduce targeted mutations (or create insertions and deletions in the BpsA gene).
We employ High-throughput screening methods to assess the ability of each BpsA variant to accept the phosphopantetheine group from non-cognate PPTases. This screening techniques might involve colorimetric assays, fluorescence-based assays, or other enzymatic activity tests that can effectively measure activation.
Our most promising variants are subjected to more rounds of mutagenesis and screening to achieve cumulative improvements in the interaction between BpsA and non-cognate PPTases.
BpsA-PPTase Interaction
Since we are interested in BpsA-PPTase Interaction, let us have a closer look at some key amino acid residues involved. The interaction between BpsA and PPTases,facilitated by specific amino acid residues at the active site is of interest. The serine residue that undergoes 4'-phosphopantetheinylation is the most critical site in BpsA for activation. Surrounding hydrophobic residues, charge-contributing residues, and flexible linker regions also play significant roles in the interaction. BpsA evolved, hence undergoes structural changes. We are introducing mutations that can lead to significant changes in the protein structure. The changes may occur in the primary sequence of amino acids, which do affect the higher levels of protein structure—secondary, tertiary, and quaternary structures. Indeed, such structural modifications can enhance the catalytic efficiency, substrate specificity, stability, and solubility of BpsA.
Near the active site, we modify residues, through our directed evolution process we enhance the catalytic efficiency of BpsA. Changes that improve the orientation or proximity of catalytic residues can increase the rate of enzymatic reactions. Furthermore, said structural modifications can also broaden or alter the substrate specificity of BpsA, enabling the production of new polyketide chains that were previously inaccessible. At this point we are making sufficient progress since the ability to enhance the activation of BpsA by non-cognate PPTases may lead to the production of novel bioactive compounds (in engineered biosynthetic pathways). Such compounds could indeed have properties opportune for treating symptoms or underlying causes of ASD, improving the life quality of the ASD patient. They have the potential to modulate neural function, improve synaptic connectivity, or correct imbalances in neurotransmitter systems.
Validation
The compounds in question - produced through these engineered pathways - can be screened for biological activities that are beneficial, such as neuroprotection, modulation of synaptic function, or inflammation reduction. When we employ High-throughput screening assays we identify compounds with the desired bioactivity, which can then be , once more optimized through additional rounds of directed evolution and chemical modification.
Integrating the evolved BpsA variants into biosynthetic pathways, we could in theory create novel compounds while engineering our pathways making use of microbial hosts, such as bacteria or yeast, to produce the compounds in large quantities for additional testing and development.
To validate the functionality of the evolved BpsA variants, a series of in vitro and in vivo assays must be conducted. In vitro assays could include enzyme activity measurements, binding affinity tests, and stability assessments. Regarding vivo assays ,expressing the evolved BpsA in microbial hosts and evaluating the production of the desired compounds is key. Structural analysis techniques,(X-ray,NMR spectroscopy, cryo-EM,..) help analyse changes induced, revealing precise alterations in the protein structure (that enhance its interaction with non-cognate PPTases and improve its catalytic efficiency). Biochemical assays help us measure the kinetic parameters of the evolved BpsA variants, such as the Michaelis constant (Km) and the maximum reaction rate (Vmax). These parameters can provide rich information on the efficiency and specificity of the evolved enzymes, guiding increased optimization efforts.
Molecular Mechanisms
One of the challenges in developing evolved enzymes for therapeutic applications is the potential for off-target effects. These effects can arise from unintended interactions with other proteins or pathways in the host organism. To mitigate these risks, extensive in-depth screening and validation processes are necessary to ensure the specificity and safety of the evolved enzymes. Ideally we want to scale up the production process (of evolved BpsA variants for industrial or therapeutic use) and this presents several challenges. such challenges include optimizing the expression and purification processes, ensuring the stability and activity of the enzymes during large-scale production, and of course, minimizing production costs where possible. Keeping track of recent advances in bioprocessing technologies and microbial engineering can help us address these problems at hand. Demonstrating the efficacy and specificity of the enzymes, will help us ensure consistent production quality. We should not overlook if these enzymes can be used to produce antibiotics, anticancer agents, or even immunosuppressants. We can integrate predictive biosynthesis inside our roadmap.
BpsA (being a non-ribosomal peptide synthetase) has a certain function responsible for synthesizing the blue pigment indigoidine. The enzyme operates through a modular architecture, where each module is responsible for specific steps in the biosynthetic pathway.
We know the carrier protein domain of BpsA plays a crucial role in this process, requiring activation by PPTases to function , in effect, and we know the carrier protein domain of BpsA contains a conserved serine residue that is the site of post-translational modification. This serine residue especially is of good use for us, since it is crucial for the attachment of the 4'-phosphopantetheine group, which is necessary for the enzyme's activity. The carrier protein domain acts as a scaffold, facilitating the transfer of intermediates between different catalytic sites within the enzyme.
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