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J Bacteriol, April 1998, p. 1814-1821, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5'-Phosphate Biosynthesis in Escherichia coli K-12

Yong Yang, Ho-Ching Tiffany Tsui, Tsz-Kwong Man, and Malcolm E. Winkler*

Department of Microbiology and Molecular Genetics, University of Texas--- Houston Medical School, Houston, Texas 77030-1501

Received 9 December 1997/Accepted 4 February 1998

pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5'-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY+ overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette into pdxY and crossed the resulting pdxY::Omega Kanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts of pdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6 vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show that pdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon. pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in the tyrS-pdxY intercistronic region.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas---Houston Medical School, 6431 Fannin; JFB 1.765, Houston, TX 77030-1501. Phone: (713) 500-5461. Fax: (713) 500-5499. E-mail: mwinkler{at}utmmg.med.uth.tmc.edu.




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