Bioretrosynthetic construction of a didanosine biosynthetic pathway

Concatenation of engineered biocatalysts into multistep pathways dramatically increases their utility, but development of generalizable assembly methods remains a significant challenge. Herein we evaluate ‘bioretrosynthesis’, which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2,3-dideoxyinosine). Applying both directed evolution and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and 3-fold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase, but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase

Bioretrosynthetic construction of a didanosine biosynthetic pathway

Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2′,3′-dideoxyinosine). Applying both directed evolution– and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase

Molecular Differences between a Mutase and a Phosphatase: Investigations of the Activation Step in <i>Bacillus cereus</i> Phosphopentomutase

Prokaryotic phosphopentomutases (PPMs) are di-Mn²⁺ enzymes that catalyze the interconversion of α-d-ribose 5-phosphate and α-d-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of <i>Bacillus cereus</i> PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, <i>B. cereus</i> PPM engages substrates when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in a catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations of the PPM^T85E phosphomimetic variant and the neutral corollary PPM^T85Q determined that the side chain of Lys-240 underwent a change in conformation in response to active site charge, which modestly influenced the affinity for the small molecule activator α-d-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated <i>B. cereus</i> PPM revealed a dramatic change in the interdomain angle and a new hydrogen bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to α-d-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPM^D156A variant even under stringent activation conditions. These results permit a proposal for activation of PPM and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase

Crystallization and preliminary X-ray analysis of a phosphopentomutase from Bacillus cereus

Two crystal forms of an Mn2+-dependent phosphopentomutase were identified from chemically distinct conditions by sparse-matrix screening with and without the inclusion of 50 mM Mn2+. The crystals identified in the presence of Mn2+ were of dramatically better diffraction quality than those identified in the absence of added Mn2+