Oberhardt, Matthew A.Zarecki, RaphyReshef, LeahXia, FangfangDuran Frigola, MiquelSchreiber, RachelHenry, Christopher S.Ben-Tal, NirDwyer, Daniel J.Gophna, UriRuppin, Eytan2016-06-062016-06-062016Oberhardt MA, Zarecki R, Reshef L, Xia F, Duran-Frigola M, Schreiber R et al. Systems-wide prediction of enzyme promiscuity reveals a new underground alternative route for pyridoxal 5'-phosphate production in E. coli. PLoS computational biology. 2016; 12(1): e1004705. DOI 10.1371/journal.pcbi.10047051553-734Xhttp://hdl.handle.net/10230/26855Recent 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.application/pdfengThis is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the/nCreative Commons CC0 public domain dedicationEnzimsFilogeniaSystems-wide prediction of enzyme promiscuity reveals a new underground alternative route for pyridoxal 5'-phosphate production in E. coliinfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1371/journal.pcbi.1004705info:eu-repo/semantics/openAccess