title: RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.
abstract: Pyridoxal 5'-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA. IMPORTANCE External stresses that disrupt metabolic components can perturb cellular functions and affect growth. A similar consequence is expected if endogenously generated metabolites are reactive and persist in the cellular environment. Here we show that the metabolic intermediate 2-aminoacrylate (2AA) causes significant cellular damage if allowed to accumulate aberrantly. Furthermore, we show that the widely conserved protein RidA prevents this accumulation by facilitating conversion of 2AA to a stable metabolite. This work demonstrates that the reactive metabolite 2AA, previously considered innocuous in the cell due to a short half-life in aqueous solution, can survive in the cellular environment long enough to cause damage. This work provides insights into the roles and persistence of reactive metabolites in vivo and shows that the RidA family of proteins is able to prevent damage caused by a reactive intermediate that is created as a consequence of PLP-dependent chemistry.
authors: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Jennifer A
authors: JA
authors: Lambrecht
authors: George E
authors: GE
authors: Schmitz
authors: Diana M
authors: DM
authors: Downs
mesh_terms: D000179:Acrylates / Q000378:metabolism / Q000633:toxicity; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D017353:Gene Deletion; D012695:L-Serine Dehydratase / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D000179:Acrylates; D001426:Bacterial Proteins; D003067:Coenzymes; C116617:YjgF protein, Bacteria; D011732:Pyridoxal Phosphate; D012695:L-Serine Dehydratase
references: J Bacteriol. 1985 Dec;164(3):1350-2
references: 3934143
references: J Biol Chem. 1981 Jul 10;256(13):6917-26
references: 7240253
references: Biochemistry. 1976 Oct 19;15(21):4718-23
references: 974085
references: J Biochem. 1997 Apr;121(4):637-41
references: 9163511
references: J Biol Chem. 2012 Jun 8;287(24):20369-81
references: 22505717
references: J Bacteriol. 1998 May;180(9):2502-6
references: 9573204
references: J Bacteriol. 2008 Apr;190(8):3057-62
references: 18296521
references: J Bacteriol. 2008 Dec;190(24):7957-65
references: 18931110
references: J Bacteriol. 1974 Oct;120(1):559-61
references: 4370904
references: Biochemistry. 1974 Sep 10;13(19):3859-63
references: 4472160
references: ACS Chem Biol. 2013 Jan 18;8(1):242-8
references: 23101964
references: J Bacteriol. 2005 Nov;187(22):7866-9
references: 16267312
references: J Biol Chem. 1965 Dec;240(12):4703-9
references: 5321308
references: J Biol Chem. 2010 Nov 5;285(45):34401-7
references: 20817725
references: Eur J Biochem. 1971 Aug 16;21(3):438-46
references: 5569609
references: J Biol Chem. 1970 Oct 25;245(20):5416-23
references: 4319241
references: J Bacteriol. 2010 Aug;192(16):4089-102
references: 20400551
references: J Bacteriol. 2002 Nov;184(21):6056-9
references: 12374840
references: Eur J Biochem. 1978 Nov 15;91(2):527-32
references: 365527
references: Mol Cell Biol. 2000 Oct;20(20):7784-97
references: 11003673
references: Biochemistry. 1979 May 1;18(9):1751-5
references: 435483
references: Planta. 2006 Dec;225(1):89-102
references: 16845531
references: J Biol Chem. 2012 Jan 27;287(5):3454-61
references: 22094463
references: J Bacteriol. 2004 Feb;186(3):803-10
references: 14729707
references: Biochem J. 1986 May 15;236(1):193-200
references: 2431676
references: J Bacteriol. 1998 Dec;180(24):6519-28
references: 9851994
references: J Bacteriol. 2006 Oct;188(19):6786-92
references: 16980480
references: J Biol Chem. 1976 Apr 10;251(7):2023-9
references: 773932
references: Biochemistry. 1964 Jul;3:885-93
references: 14214072
references: Anal Biochem. 1973 Jan;51(1):67-79
references: 4569372
references: Biochemistry. 2001 Jun 26;40(25):7453-63
references: 11412098
references: J Bacteriol. 2000 Feb;182(3):573-80
references: 10633088
references: Biochemistry. 1969 Nov;8(11):4477-82
references: 4311034
references: J Biol Chem. 1956 Jan;218(1):97-106
references: 13278318
references: Biochemistry. 2003 Jan 14;42(1):80-9
references: 12515541
references: Proc Natl Acad Sci U S A. 1964 Mar;51:382-9
references: 14171448
references: Eur J Biochem. 2004 Aug;271(15):3248-54
references: 15265044
references: Genes Cells. 2001 Jun;6(6):507-17
references: 11442631
references: Arch Microbiol. 2000 Oct;174(4):265-72
references: 11081795
references: PLoS One. 2012;7(8):e43082
references: 22900093
references: Appl Environ Microbiol. 2005 Jul;71(7):4149-52
references: 16000837
references: FEBS Lett. 1971 Dec 15;19(3):225-228
references: 11946217
references: J Biol Chem. 1984 Sep 10;259(17):10669-74
references: 6432787
references: Biochemistry. 1984 Oct 23;23(22):5188-94
references: 6439236
references: Biochemistry. 1979 Aug 7;18(16):3557-63
references: 383145
references: Biochemistry. 1982 Aug 31;21(18):4387-93
references: 6812625
references: Arthritis Res. 2002;4(4):266-73
references: 12106498
references: FEMS Microbiol Lett. 1998 Feb 15;159(2):261-6
references: 9503620
references: Biochemistry. 1969 Dec;8(12):5016-21
references: 5365791
references: J Bacteriol. 1957 Jan;73(1):105-12
references: 13405870
references: Annu Rev Biochem. 1984;53:493-535
references: 6433782
references: Arch Microbiol. 1998 Dec;171(1):1-5
references: 9871012
references: Biochemistry. 1982 Aug 31;21(18):4377-86
references: 6812624
references: J Bacteriol. 1971 Feb;105(2):519-22
references: 5541528
references: J Biol Chem. 1974 Mar 10;249(5):1519-24
references: 4817757
references: FEMS Microbiol Lett. 2009 Sep;298(1):93-8
references: 19594622
references: Microbiol Rev. 1979 Jun;43(2):260-96
references: 390357
references: J Bacteriol. 1971 Dec;108(3):1277-83
references: 4945194
references: Annu Rev Biochem. 2008;77:755-76
references: 18173371
references: Biochemistry. 1984 Oct 23;23(22):5195-201
references: 6439237