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Journal of Bacteriology, July 2009, p. 4158-4165, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00416-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

6-Pyruvoyltetrahydropterin Synthase Paralogs Replace the Folate Synthesis Enzyme Dihydroneopterin Aldolase in Diverse Bacteria ^,

Anne Pribat,¹ Linda Jeanguenin,¹ Aurora Lara-Núñez,² Michael J. Ziemak,¹ John E. Hyde,³ Valérie de Crécy-Lagard,⁴ and Andrew D. Hanson^1*

Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611,¹ Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611,² Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom,³ Microbiology and Cell Science Department, University of Florida, Gainesville, Florida 32611⁴

Received 26 March 2009/ Accepted 19 April 2009

Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.

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* Corresponding author. Mailing address: University of Florida, Horticultural Sciences Department, P.O. Box 110690, Gainesville, FL 32611. Phone: (352) 392-1928. Fax: (352) 392-5653. E-mail: adha{at}ufl.edu

Published ahead of print on 24 April 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, July 2009, p. 4158-4165, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00416-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.