Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5'-Phosphate Production in E. coli

PLoS Comput Biol. 2016 Jan 28;12(1):e1004705. doi: 10.1371/journal.pcbi.1004705. eCollection 2016 Jan.


Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbohydrate Dehydrogenases / genetics
  • Carbohydrate Dehydrogenases / metabolism
  • Computational Biology / methods*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Models, Molecular
  • Pyridoxal Phosphate / metabolism*


  • Escherichia coli Proteins
  • ThiG protein, E coli
  • Pyridoxal Phosphate
  • Carbohydrate Dehydrogenases
  • pdxB protein, E coli

Grants and funding

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding agencies: (MO) Whitaker Foundation (Whitaker International Scholars Program) ( (MO) Dan David Fellowship ( (ER) European Union FP7 INFECT project ( ERA-Net Plant project ( (ER) I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant No 41/11) ( (UG) McDonnell foundation ( (UG) German-Israeli Project Cooperation (DIP) ( (MD) Spanish FPU grant ( (MD) FEBS short term fellowship ( (NBT) Grant No. 1775/12 of the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation