Enzymatic pathways have evolved over billions of years to carry out essential cellular processes and ensure the survival of their host species. These reaction pathways rely on the interconnectedness of multiple enzymes and substrate, encouraging cross-talk and, at times, competition. In many cases, enzymes require the assistance of a diffusible secondary biomolecule, known as a cofactor, to participate in catalytic reactions. This network of reactions is unfavorable when trying to optimize the production of a specific product. In order to circumvent surrounding reactions, researchers have been engineering orthogonal enzymatic pathways that operate independently from endogenous reactions within a cell. Orthogonal pathways can be created by utilizing biomimetics molecules; most enzymes have not naturally evolved affinity and activity with these are non-canonical cofactors.
Nicotinamide adenine dinucleotide (NAD(H) and nicotinamide adenine dinucleotide 2’-phosphate (NADP(H)) are vital cofactors that participate in redox reactions within cells. NAD(P)(H) have been the target of enzymatic research for several decades due to their extensive involvement in reactions across species and their utility in the biotechnology industry. Creation of orthogonal pathways dependent on NAD(P)(H) analogs has massive potential in various industries, such as biofuels and biopharmaceuticals. Nicotinamide mononucleotide (NMN(H)) is a precursor molecule in the biosynthesis of NAD(H); it currently exists within cells but, in general, does not participate as a cofactor. Nicotinamide adenine dinucleotide 3’-phosphate (3’-NADP(H)) is another analog that closely resembles NAD(P)(H) for which most enzymes have not evolved natural affinity and activity.
Computation and structural inspection techniques were used in an attempt to engineer formate dehydrogenase from Candida boidinii (CbFDH) for activity with the non-canonical cofactors NMN(H) and 3’-NADP(H). Amino acid positions proximal to the NAD(H) binding site were input into a PyRosetta algorithm, which then outputted a list of recommended mutations ranked by their Rosetta energy scores. Structural alignment and visual inspection were also used to design mutations. The mutations were recombinantly expressed, and the purified enzymes were assays with NAD+, NADP+, NMN+ and 3’-NADP+. None of the designed single mutations led to CbFDH activity gain with NMN+ to any meaningful degree; however, various mutations led to the removal of NAD+ activity. A strength of PyRosetta was identifying key mutations that would lead to activity removal. The single mutants D195A and D195G attained the largest specific initial rates with 3’-NADP+ under the screening assay conditions. Kinetics parameters of a simplified ordered bi bi model were calculated for these mutants. Double mutants were created in an attempt to further enhance activity. The double mutations resulted in decreased activity but enhanced the specificity for 3’-NADP+ over NAD+.
To complete the 3’-NADP(H) enzymatic cycle, a non-specific cofactor oxidizer, xenobiotic reductase A (XenA), was expressed and assayed with 3’-NADPH. It was found that XenA is able to oxidize 3’-NADPH back to 3’-NADP+. The avirulence factor AvrRxo1 from a rice plant pathogen was explored since it specifically catalyzes NAD+ phosphorylation at the 3’ position. The AvrRxo1 gene was expressed in LysY/IQ competent E. coli cells and it was found that the presence of AvrRxo1 caused a longer lag phase, but the bacteria were later able to recover. Co-expressing AvrRxo1, XenA, and D195A CbFDH has the potential to create an orthogonal pathway depending on biosynthesized 3’-NADP(H) in vivo. Another in vivo non-canonical cofactor source is NAD(H)-capped RNA, which have recently captured researchers’ attention. NAD+-RNA was synthesized using the polymerase chain reaction, and it was shown that D195A and D195G CbFDH were able to reduce the NAD+ cap.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/g74d-c421 |
| Date | January 2023 |
| Creators | Vainstein, Salomon |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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