Structure of PqsD, a Pseudomonas quinolone signal biosynthetic enzyme, in complex with anthranilate.
Pseudomonas quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, is an intercellular alkyl quinolone signaling molecule produced by the opportunistic pathogen Pseudomonas aeruginosa. Alkyl quinolone signaling is an atypical system that, in P. aeruginosa, controls the expression of numerous virulence factors. PQS is synthesized from the tryptophan pathway intermediate, anthranilate, which is derived either from the kynurenine pathway or from an alkyl quinolone specific anthranilate synthase encoded by phnAB. Anthranilate is converted to PQS by the enzymes encoded by the pqsABCDE operon and pqsH. PqsA forms an activated anthraniloyl-CoA thioester that shuttles anthranilate to the PqsD active site where it is transferred to Cys112 of PqsD. In the only biochemically characterized reaction, a condensation then occurs between anthraniloyl-PqsD and malonyl-CoA or malonyl-ACP, a second PqsD substrate, forming 2,4-dihydroxyquinoline (DHQ). The role PqsD plays in the biosynthesis of other alkyl quinolones, such as PQS, is unclear, though it has been reported to be required for their production. No evidence exists that DHQ is a PQS precursor, however. Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme, including that of the PqsD-anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical approximately 170-residue alphabetaalphabetaalpha domains. The structures show anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of an approximately 15 A long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive, although it still reversibly binds anthraniloyl-CoA. The covalent complex between anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.
Bera
Asim K
AK
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Atanasova
Vesna
V
Robinson
Howard
H
Eisenstein
Edward
E
Coleman
James P
JP
Pesci
Everett C
EC
Parsons
James F
JF
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015363:Quinolones / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000737:chemistry*
C407944:2-heptyl-3-hydroxy-4-quinolone; D001426:Bacterial Proteins; D005227:Fatty Acids; D015363:Quinolones; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase
J Bacteriol. 2008 Feb;190(4):1247-55
18083812
J Bacteriol. 2005 Jul;187(13):4372-80
15968046
Acta Crystallogr D Biol Crystallogr. 2001 Jan;57(Pt 1):122-33
11134934
Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11229-34
10500159
Protein Eng. 2001 Dec;14(12):993-1000
11809930
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Biochemistry. 2004 Oct 5;43(39):12427-35
15449932
Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):32-41
17164524
Biochemistry. 2000 Feb 8;39(5):890-902
10653632
Clin Rev Allergy Immunol. 2008 Dec;35(3):124-34
18509765
J Bacteriol. 2008 Nov;190(21):7043-51
18776012
J Bacteriol. 2005 Jun;187(11):3630-5
15901684
Mol Microbiol. 2005 Feb;55(4):998-1014
15686549
J Bacteriol. 2002 Dec;184(23):6472-80
12426334
J Mol Biol. 1997 Oct 31;273(3):714-28
9402066
J Antimicrob Chemother. 2003 Oct;52(4):668-74
12951354
Structure. 2000 Feb 15;8(2):185-95
10673437
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Acta Crystallogr D Biol Crystallogr. 2008 Jan;64(Pt 1):61-9
18094468
Chem Biol. 2008 Apr;15(4):402-12
18420147
Chem Biol. 2006 Jul;13(7):701-10
16873018
Nat Prod Rep. 2003 Feb;20(1):79-110
12636085
J Mol Biol. 2001 Mar 16;307(1):341-56
11243824
Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1339-44
14739337
Curr Opin Microbiol. 2003 Feb;6(1):56-60
12615220
J Biol Chem. 2008 Oct 24;283(43):28788-94
18728009
Biochemistry. 2007 Feb 20;46(7):1821-8
17253782
Acta Crystallogr D Biol Crystallogr. 1999 Oct;55(Pt 10):1718-25
10531521
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14613-8
11724939
Mol Microbiol. 2006 Dec;62(6):1689-99
17083468
J Biol Chem. 2000 Dec 15;275(50):39640-6
11006298
Emerg Infect Dis. 1998 Oct-Dec;4(4):551-60
9866731
Curr Opin Microbiol. 2009 Feb;12(1):61-6
19168385
Anal Bioanal Chem. 2007 Jan;387(2):469-79
17139483
Biochemistry. 2008 May 13;47(19):5281-9
18416536
Protein Expr Purif. 2005 May;41(1):207-34
15915565
Nat Struct Biol. 1999 Aug;6(8):775-84
10426957