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Journal of Bacteriology, May 2003, p. 2848-2855, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2848-2855.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD from Pseudomonas aeruginosa

David F. Ackerley,{dagger} Tom T. Caradoc-Davies, and Iain L. Lamont*

Department of Biochemistry, University of Otago, Dunedin, New Zealand

Received 21 October 2002/ Accepted 3 January 2003

Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdinePAO, which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. L-Threonine, one of the amino acid residues in pyoverdinePAO, was an effective substrate for the recombinant protein in ATP-PPi exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including D-threonine, L-serine, and L-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases.

* Corresponding author. Mailing address: Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand. Phone: 64 3 479 7869. Fax: 64 3 479 7866. E-mail: iain.lamont{at}stonebow.otago.ac.nz.

{dagger} Present address: Department of Microbiology and Immunology, Stanford University, Stanford, Calif.

Journal of Bacteriology, May 2003, p. 2848-2855, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2848-2855.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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