JB Accepts, published online ahead of print on 6 November 2009

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J. Bacteriol. doi:10.1128/JB.01198-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

FolX and FolM Are Essential for Tetrahydromonapterin Synthesis in Escherichia coli and Pseudomonas aeruginosa

Anne Pribat, Ian K. Blaby, Aurora Lara-Núñez, Jesse F. Gregory III, Valérie de Crécy-Lagard^*, and Andrew D. Hanson^*

Horticultural Sciences Department, Microbiology and Cell Science Department, and Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611

^* To whom correspondence should be addressed. Email: vcrecy{at}ufl.edu. adha{at}ufl.edu.

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis, via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were experimentally tested first in E. coli, which lacks PhhA and in which expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase PhhB rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The tyrA strain was, as expected, prototrophic for tyrosine whereas the tyrA folX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end-product of pterin biosynthesis.