title: An in vivo metabolic approach for deciphering the product specificity of glycerate kinase proves that both E. coli's glycerate kinases generate 2-phosphoglycerate.
abstract: Apart from addressing humanity's growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli's phosphoglycerate mutases, we divide its central metabolism into an 'upper' and 'lower' metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli's enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions.
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel.
authors.forename: Lior
authors.initials: L
authors.lastname: Zelcbuch
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel; Department of Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Manuel
authors.initials: M
authors.lastname: Razo-Mejia
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel.
authors.forename: Elad
authors.initials: E
authors.lastname: Herz
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel.
authors.forename: Sagit
authors.initials: S
authors.lastname: Yahav
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel.
authors.forename: Niv
authors.initials: N
authors.lastname: Antonovsky
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel.
authors.forename: Hagar
authors.initials: H
authors.lastname: Kroytoro
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel.
authors.forename: Ron
authors.initials: R
authors.lastname: Milo
authors.affiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot Israel; Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
authors.forename: Arren
authors.initials: A
authors.lastname: Bar-Even
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D005988:Glyceric Acids / Q000378:metabolism*; D060847:Metabolic Engineering; D010736:Phosphoglycerate Mutase / Q000172:deficiency / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005988:Glyceric Acids; C008885:2-phosphoglycerate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020670:glycerate kinase; D010736:Phosphoglycerate Mutase
references.citation: Biochemistry. 2005 Apr 26;44(16):6371-82
references.pmid: 15835926
references.citation: J Bacteriol. 1977 Nov;132(2):398-410
references.pmid: 410789
references.citation: PLoS One. 2010;5(10):e13576
references.pmid: 21187861
references.citation: Mol Syst Biol. 2011;7:535
references.pmid: 21988831
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D770-5
references.pmid: 22064852
references.citation: Nat Chem Biol. 2012 Nov;8(11):926-32
references.pmid: 23042035
references.citation: Nucleic Acids Res. 2013 May;41(9):e98
references.pmid: 23470993
references.citation: PLoS Genet. 2013;9(7):e1003665
references.pmid: 23935519
references.citation: PLoS Comput Biol. 2014 Feb;10(2):e1003483
references.pmid: 24586134
references.citation: Appl Microbiol Biotechnol. 2014 Feb;98(3):1165-74
references.pmid: 23666480
references.citation: Trends Plant Sci. 2010 Jun;15(6):330-6
references.pmid: 20403720
references.citation: Mol Syst Biol. 2006;2:2006.0008
references.pmid: 16738554
references.citation: Nat Protoc. 2007;2(4):924-32
references.pmid: 17446874
references.citation: Curr Protoc Mol Biol. 2007 Jul;Chapter 1:Unit 1.17
references.pmid: 18265391
references.citation: FEBS Lett. 2008 Sep 3;582(20):3025-8
references.pmid: 18675808
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 24;106(8):2874-9
references.pmid: 19196979
references.citation: Biochemistry. 2014 Jul 1;53(25):4161-8
references.pmid: 24914732
references.citation: J Bacteriol. 1999 Dec;181(24):7479-84
references.pmid: 10601204
references.citation: Structure. 2003 Sep;11(9):1141-50
references.pmid: 12962632
references.citation: Appl Environ Microbiol. 2004 Feb;70(2):1238-41
references.pmid: 14766613
references.citation: J Biol Chem. 1966 Feb 10;241(3):568-72
references.pmid: 5325263
references.citation: J Bacteriol. 1969 Mar;97(3):1227-33
references.pmid: 4887503
references.citation: Biochemistry. 1998 Oct 13;37(41):14369-75
references.pmid: 9772162
references.citation: Gene. 1995 May 26;158(1):9-14
references.pmid: 7789817
references.citation: J Bacteriol. 1981 Sep;147(3):805-19
references.pmid: 6115851
references.citation: J Bacteriol. 1980 Oct;144(1):114-23
references.pmid: 6998942
references.citation: Plant Cell. 2005 Aug;17(8):2413-20
references.pmid: 15980259