Structure of the epd (gapB) gene (drawn to scale) at 66.17 min in the E. coli K-12 chromosome (1, 15, 21). epd (gapB) is surrounded by and oriented in the same direction as yggC, whose function is unknown, and pgk, which encodes the glycolytic enzyme phosphoglycerate kinase (1). Promoter mapping studies to be published elsewhere locate the promoters for epd (gapB) (P_epd) and pgk (P_pgk) in the regions indicated by bars. The location of the epd::Ω(Cm^r) insertion mutation constructed in this study (Table 1) is indicated. The epd::Ω(Cm^r) insertion mutation is upstream from the P_pgk promoter region and does not interfere with transcription of the pgk gene (data not shown).

Pathway for PLP and PMP coenzyme biosynthesis in E. coli K-12. Enzymes that catalyze the steps in the pathway are indicated by their genetic symbols and are boxed. Branch 1 takes E4P to 4PHT, and branch 2 provides DXP, which is condensed with 4PHT to form PNP. PNP is oxidized to the active coenzyme PLP, which can be converted to PMP by transaminases. Oxidation of E4P to 4PE is the first step of branch 1 and is catalyzed by the E4P dehydrogenase activities of the GapA and Epd (GapB) enzymes. See text for details.

HPLC chromatograms of B₆ vitamers extracted from strains TX3470 (gapA⁺ epd⁺ parent) (top), TX3481 [(gapA⁺ epd::Ω(Cm^r)] (middle), and TX3484 (gapA1 epd⁺) (bottom). Strains were grown on plates as described in footnote a to Table 3, and B₆ vitamers were partially purified by metaphosphoric acid and dichloromethane extractions and were resolved by reverse-phase, ion-pair HPLC on an Ultremex 3 C₁₈ column (150 by 4.6 mm) (Phenomenex, Inc.) fitted with a guard column (30 by 4.6 mm) at 0.5 ml per min as described elsewhere (31). The tracings show the fluorescence intensity (excitation at 328 nm; emission at 393 nm) of postcolumn adducts between the B₆ vitamers and sodium bisulfite (31). The elution positions and fluorescence yields of the six B₆ vitamers (PLP, PMP, PNP, PL, PM, PN) were determined by using pure compounds as standards (data not shown). Only PLP and PMP (top panel) were detected in extracts of E. coli K-12. Two peaks that are not B₆ vitamers were routinely detected (top panel) and were used along with protein amounts of the cells before extraction to normalize PLP and PMP amounts in different preparations. Amounts of PLP and PMP were determined (Table 3) by integration of peaks and comparison of areas with those of the PLP and PMP standard curves, which were linear over this range of concentrations. The fluorescence yield of the PMP-bisulfite adduct was approximately 5.6-fold greater than that of the PLP-bisulfite adduct (data not shown). This difference accounts for the fact that PLP peaks, which are apparently smaller than PMP peaks, actually correspond to more PLP than PMP (Fig. 3; Table 3).