Purification and characterization of OleA from Xanthomonas campestris and demonstration of a non-decarboxylative Claisen condensation reaction.
OleA catalyzes the condensation of fatty acyl groups in the first step of bacterial long-chain olefin biosynthesis, but the mechanism of the condensation reaction is controversial. In this study, OleA from Xanthomonas campestris was expressed in Escherichia coli and purified to homogeneity. The purified protein was shown to be active with fatty acyl-CoA substrates that ranged from C(8) to C(16) in length. With limiting myristoyl-CoA (C(14)), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [(14)C]myristoyl-CoA, the other products were identified as myristic acid, 2-myristoylmyristic acid, and 14-heptacosanone. 2-Myristoylmyristic acid was indicated to be the physiologically relevant product of OleA in several ways. First, 2-myristoylmyristic acid was the major condensed product in short incubations, but over time, it decreased with the concomitant increase of 14-heptacosanone. Second, synthetic 2-myristoylmyristic acid showed similar decarboxylation kinetics in the absence of OleA. Third, 2-myristoylmyristic acid was shown to be reactive with purified OleC and OleD to generate the olefin 14-heptacosene, a product seen in previous in vivo studies. The decarboxylation product, 14-heptacosanone, did not react with OleC and OleD to produce any demonstrable product. Substantial hydrolysis of fatty acyl-CoA substrates to the corresponding fatty acids was observed, but it is currently unclear if this occurs in vivo. In total, these data are consistent with OleA catalyzing a non-decarboxylative Claisen condensation reaction in the first step of the olefin biosynthetic pathway previously found to be present in at least 70 different bacterial strains.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108, USA.
Janice A
JA
Frias
Jack E
JE
Richman
Jasmine S
JS
Erickson
Lawrence P
LP
Wackett
D000475:Alkenes / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003065:Coenzyme A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005227:Fatty Acids / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016959:Xanthomonas campestris / Q000201:enzymology*
D000475:Alkenes; D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D003065:Coenzyme A
Arch Biochem Biophys. 1958 Apr;74(2):443-50
13534673
Science. 2010 Jul 30;329(5991):559-62
20671186
Biochemistry. 1969 Jan;8(1):394-404
5777337
Appl Environ Microbiol. 2010 Feb;76(4):1212-23
20038703
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
9254694
Appl Environ Microbiol. 2010 Jun;76(12):3842-9
20418444
J Bacteriol. 1967 Aug;94(2):349-58
6039358
Anal Biochem. 2010 Jan 1;396(1):96-102
19706283
Appl Environ Microbiol. 2010 Jun;76(12):3850-62
20418421
Nat Prod Rep. 2002 Oct;19(5):581-96
12430724
FEMS Microbiol Lett. 2007 Jan;266(2):138-43
17233723
Biochemistry. 1969 Mar;8(3):953-9
5781029
J Biol Chem. 1988 Sep 25;263(27):13564-71
2901416
J Biol Chem. 1987 Jan 5;262(1):82-9
2878927
Structure. 2000 Feb 15;8(2):185-95
10673437
Biochemistry. 1969 May;8(5):1913-8
5785213
Nature. 2004 Mar 25;428(6981):441-5
15042094
J Org Chem. 2004 Oct 1;69(20):6528-32
15387574
J Biol Chem. 2004 Feb 20;279(8):6730-6
14625287
J Biol Chem. 2009 Jul 17;284(29):19255-64
19436070
Appl Microbiol Biotechnol. 2009 Oct;84(6):1003-19
19669755
Appl Environ Microbiol. 2009 Mar;75(6):1774-7
19168653
Biochemistry. 1969 Aug;8(8):3317-24
4390164
Trends Biochem Sci. 2006 Jan;31(1):64-71
16356722
Biochemistry. 1970 Apr 28;9(9):1893-8
5445330
J Biol Chem. 1975 Apr 25;250(8):3108-16
164460
Biochim Biophys Acta. 1995 Oct 5;1258(3):288-96
7548199
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Sep 1;66(Pt 9):1108-10
20823539