Investigating the promiscuous roles of Pseudomonas putida GlyA-II and ThrB in a novel PLP biosynthetic pathway

Simmons, Cat

Undergraduate Honors Thesis

Investigating the promiscuous roles of Pseudomonas putida GlyA-II and ThrB in a novel PLP biosynthetic pathway Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/b2773x10h

Abstract

The patchwork hypothesis posits how new metabolic pathways emerge via the recruitment of unspecialized or general-purpose enzymatic reactions (Jensen, 1976). These newly established pathways may be referred to as protopathways (PPs). A protopathway consists of a patched-together set of promiscuous activities, or enzymatic reactions that are not included in normal metabolism. In all known 𝛾-proteobacteria, erythronate 4-phosphate dehydrogenase (PdxB) functions in the pyridoxal 5’-phosphate (PLP) pathway. In 2019, Kim et al. revealed that when pdxB is deleted in Escherichia coli, after 130-150 generations, the evolved mutant reaches a near wild-type growth rate. These researchers elucidated a protopathway utilized by this evolved ΔpdxB E. coli strain to bypass the obstruction in the PLP biosynthetic pathway. When the same evolution experiment was performed on ΔpdxB Pseudomonas putida KT2440, the strain regained a near wild-type growth rate after only 10 population doublings. Sequencing of 4 independently evolved ΔpdxB P. putida strains each revealed a different mutation in glyA-II, encoding serine hydroxymethyltransferase. E. coli GlyA catalyzes a promiscuous reaction that is native to low-specificity L-threonine aldolase (LtaE), which also functions in an E. coli PP. Thus it was proposed that GlyA-II catalyzes LtaE’s promiscuous reaction in a P. putida PP, termed PP5. Homoserine kinase (ThrB) was proposed to act downstream of GlyA-II in PP5 because ThrB acts after LtaE in an E. coli PP. The resulting intermediate would then feed back into the main PLP pathway. Attempts were made to create glyA-II and thrB deletions in evolved ΔpdxB P. putida to determine if these mutations abolish growth, though mutants were never successfully isolated. However, enzyme assays indicated that P. putida ThrB catalyzes its proposed promiscuous reaction in vitro, and structural analysis of P. putida GlyA-II further supports the validity of PP5.

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