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.

Abstract

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*

Substances

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

Grant support

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) (http://www.whitaker.org/grants/fellows-scholars) (MO) Dan David Fellowship (http://www.dandavidprize.org/scholarship-applications) (ER) European Union FP7 INFECT project (http://www.fp7infect.eu/) ERA-Net Plant project (http://www.erapg.org/publicpage.m?key=everyone&trail=/everyone) (ER) I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant No 41/11) (www.i-core.org.il/ISF) (UG) McDonnell foundation (https://www.jsmf.org/) (UG) German-Israeli Project Cooperation (DIP) (http://www.dfg.de/en/research_funding/programmes/international_cooperation/german_israeli_cooperation/) (MD) Spanish FPU grant (http://cepima.upc.edu/positions/FPU_2013) (MD) FEBS short term fellowship (http://www.febs.org/our-activities/fellowships/febs-short-term-fellowships/guidelines-for-febs-short-term-fellowships) (NBT) Grant No. 1775/12 of the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation