PDBsum entry 4lrd

Isomerase

PDB id: 4lrd

Contents

Protein chain

391 a.a.

Ligands

TRS

GOL ×2

Metals

_MN ×3

Waters ×305

References listed in PDB file

Key reference

• Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway.
• Authors: W.R.Birmingham, C.A.Starbird, T.D.Panosian, D.P.Nannemann, T.M.Iverson, B.O.Bachmann.
• Ref.: Nat Chem Biol, 2014, 10, 392-399. [DOI no: 10.1038/nchembio.1494]
• PubMed id: 24657930

Abstract

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.