Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

A Novel Aminoacyl-tRNA Synthetase Appended Domain Can Supply the Core Synthetase with Its Amino Acid Substrate

The structural organization and functionality of aminoacyl-tRNA synthetases have been expanded through polypeptide additions to their core aminoacylation domain. We have identified a novel domain appended to the methionyl-tRNA synthetase (MetRS) of the intracellular pathogen Mycoplasma penetrans. Sequence analysis of this N-terminal region suggests the appended domain is an aminotransferase, which we demonstrate here. The aminotransferase domain of MpMetRS is capable of generating methionine from its α-keto acid analog, 2-keto-4-methylthiobutyrate (KMTB). The methionine thus produced can be subsequently attached to cognate tRNAMet in the MpMetRS aminoacylation domain. Genomic erosion in the Mycoplasma species has impaired many canonical biosynthetic pathways, causing them to rely on their host for numerous metabolites. It is still unclear if this bifunctional MetRS is a key part of pathogen life cycle or is a neutral consequence of the reductive evolution experienced by Mycoplasma species.

Human 20S proteasome activity towards fluorogenic peptides of various chain lengths

The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.