Structural and functional studies of the Escherichia coli phenylacetyl-CoA monooxygenase complex.
The utilization of phenylacetic acid (PA) in Escherichia coli occurs through a hybrid pathway that shows features of both aerobic and anaerobic metabolism. Oxygenation of the aromatic ring is performed by a multisubunit phenylacetyl-coenzyme A oxygenase complex that shares remote homology of two subunits to well studied bacterial multicomponent monooxygenases and was postulated to form a new bacterial multicomponent monooxygenase subfamily. We expressed the subunits PaaA, B, C, D, and E of the PA-CoA oxygenase and showed that PaaABC, PaaAC, and PaaBC form stable subcomplexes that can be purified. In vitro reconstitution of the oxygenase subunits showed that each of the PaaA, B, C, and E subunits are necessary for catalysis, whereas PaaD is not essential. We have determined the crystal structure of the PaaAC complex in a ligand-free form and with several CoA derivatives. We conclude that PaaAC forms a catalytic core with a monooxygenase fold with PaaA being the catalytic α subunit and PaaC, the structural β subunit. PaaAC forms heterotetramers that are organized very differently from other known multisubunit monooxygenases and lacks their conservative network of hydrogen bonds between the di-iron center and protein surface, suggesting different association with the reductase and different mechanisms of electron transport. The PaaA structure shows adaptation of the common access route to the active site for binding a CoA-bound substrate. The enzyme-substrate complex shows the orientation of the aromatic ring, which is poised for oxygenation at the ortho-position, in accordance with the expected chemistry. The PA-CoA oxygenase complex serves as a paradigm for the new subfamily multicomponent monooxygenases comprising several hundred homologs.
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Andrey M
AM
Grishin
Eunice
E
Ajamian
Limei
L
Tao
Linhua
L
Zhang
Robert
R
Menard
Miroslaw
M
Cygler
D018360:Crystallography, X-Ray; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry*; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D006899:Mixed Function Oxygenases
Proc Natl Acad Sci U S A. 2010 Aug 10;107(32):14390-5
20660314
Appl Environ Microbiol. 2006 Nov;72(11):7422-6
16997993
Biochemistry. 1997 Aug 5;36(31):9283-9
9280437
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3784-9
12640145
Proteins. 1997 Oct;29(2):141-52
9329079
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Sep 1;66(Pt 9):1045-9
20823522
Ann N Y Acad Sci. 2008 Mar;1125:82-99
18378589
Appl Environ Microbiol. 2007 Oct;73(20):6460-7
17704278
Biochemistry. 2004 Dec 28;43(51):16263-76
15610020
Acta Crystallogr D Biol Crystallogr. 1999 Jan;55(Pt 1):247-55
10089417
Cell Mol Life Sci. 2007 Feb;64(3):271-93
17221166
Appl Microbiol Biotechnol. 2009 Nov;85(2):207-28
19730850
Eur J Biochem. 2003 Jul;270(14):3047-54
12846838
Eur J Biochem. 2002 Nov;269(22):5689-99
12423369
Appl Environ Microbiol. 2006 Jun;72(6):3933-9
16751499
Nature. 1962 Aug 11;195:537-43
14039612
Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):12-21
20057044
Mol Microbiol. 2001 Mar;39(6):1434-42
11260461
Methods Enzymol. 1997;276:307-26
27754618
J Mol Evol. 2003 Apr;56(4):435-45
12664163
J Biol Chem. 2004 Jul 16;279(29):30600-10
15096510
J Biol Chem. 1998 Oct 2;273(40):25974-86
9748275
Science. 1995 May 19;268(5213):998-1004
7754395
Curr Opin Biotechnol. 2000 Jun;11(3):236-43
10851146
Appl Environ Microbiol. 2009 Feb;75(3):823-36
19074607
Acc Chem Res. 2006 Aug;39(8):558-66
16906752
Biochemistry. 1971 Jun 22;10(13):2530-6
4326771
Biochemistry. 2006 Dec 26;45(51):15392-404
17176061
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
Nature. 1993 Dec 9;366(6455):537-43
8255292
Appl Environ Microbiol. 2004 Apr;70(4):2211-9
15066815
Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19194-8
19033467
Protein Expr Purif. 2007 Apr;52(2):273-9
17049880