We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen ... Direct enzyme assays detected erythrose-4-phosphate dehydrogenase ...
JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6518-6528 0021-9193/90/116518-11$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 172, No. 11
Metabolic Relationships between Pyridoxine (Vitamin B6) and Serine Biosynthesis in Escherichia coli K-12 HON-MING LAM' AND MALCOLM E. WINKLER2*
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611,1 and Department of Microbiology, University of Texas Medical School, Houston, Texas 770302 Received 14 May 1990/Accepted 28 August 1990
We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen 1 and carbons 5, 5', and 6 of the pyridoxine ring. This pathway, which parallels the phosphorylated pathway of serine biosynthesis, is predicated on the homology between PdxB and SerA, the structural similarity between serine and 4-hydroxythreonine, and the possible involvement of SerC in pyridoxine biosynthesis. Several predictions of this hypothetical scheme were tested. Consistent with the proposed pathway, supplement inhibition patterns strongly suggest that SerA enzyme acts in an alternate pathway of pyridoxine biosynthesis in pdxB mutants. Direct enzyme assays detected erythrose-4-phosphate dehydrogenase activity in crude extracts, which again supports the proposed pathway. Chromosomal insertions in serC caused a requirement for pyridoxine, serine, and aromatic compounds, which directly verified that SerC functions in the pyridoxine biosynthetic pathway. Complementation analysis showed that pdxF and pdxC mutations reported previously are most likely alleles of serC. Growth of serC chromosomal insertion mutants on glycolaldehyde was found to occur without acquisition of second-site mutations and confirmed that pdxB and serC, but not pdxA, function in the same branch of the pyridoxine pathway. In addition, serC::mini-Mu d insertions revealed that the complex serC-aroA operon lacks internal promoters, that the amino terminus of SerC is not strictly essential for activity, and that antisense transcription occurs in the serC-aroA operon. Growth responses of pdxA, pdxB, and serC mutants to I-hydroxypyruvate, D-alanine, and glycolate could also be reconciled with the proposed pathway. Finally, the proposed scheme is consistent with previous isotope labeling data and accounts for several other observations about pyridoxine biosynthesis. Together, these physiological and biochemical analyses support the proposed pathway and an evolutionary scenario in which this branch of the pyridoxine pathway evolved from the serine pathway by gene recruitment.
Pyridoxine (vitamin B6) is the pyridine ring-containing precursor of essential coenzyme pyridoxal phosphate, which is utilized by enzymes in all phases of amino acid metabolism (7). Pyridoxine is synthesized by numerous bacteria, fungi, and higher plants, whereas the steps that convert pyridoxine into pyridoxal phosphate appear to be found in all organisms (52). Because of its importance to amino group metabolism, pyridoxine and pyridoxal phosphate biochemistry have been studied intensively for over 30 years (for reviews, see references 18, 19, 27, and 29), yet the pyridoxine biosynthetic pathway remains largely unknown. Combined genetic, physiological, and biochemical analyses using Escherichia coli B and K-12 strains suggest two likely precursors of pyridoxine. Consideration of nutritional requirements and isotope labeling patterns led Dempsey to propose that 4-hydroxythreonine undergoes a decarboxylation and then supplies nitrogen 1 and carbons 5, 5', and 6 to the pyridoxine ring (Fig. 1) (19). Recent nuclear magnetic resonance analyses by Hill and co-workers implicate D-1deoxyxylulose or a 4-oxo derivative as the donor of remaining carbons 2, 2', 3, 4, and 4' to the pyridoxine ring (28). However, the steps leading to these putative intermediates have been neither delineated nor related to functions of the mapped pyridoxine (pdx) biosynthetic genes, which fall into at least five linkage groups and are members of complex, multifunctional operons in E. coli K-12 (Lam and Winkler, unpublished results) (1, 22, 40). Recently, we reported that the pdxB and serA gene
products of E. coli K-12 are evolutionarily related (42). The serA gene encodes 3-phosphoglycerate dehydrogenase, which catalyzes the first step of the major phosphorylated pathway of serine biosynthesis (Fig. 1) (47). This analysis strongly suggests that the pdxB gene product is a 2-hydroxyacid dehydrogenase and that the branch of pyridoxine biosynthesis mediated by pdxB evolved from the phosphorylated pathway of serine biosynthesis (42). Further, the close structural similarity between serine and 4-hydroxythreonine and the possible involvement of serC-encoded phosphoserine aminotransferase in pyridoxine biosynthesis (20, 30, 44) suggest the pathway depicted in Fig. 1 (42). In this scheme, the pdxB gene product encodes erythronate-4-phosphate dehydrogenase and the SerC aminotransferase acts at a point parallel to the phosphorylated serine biosynthetic pathway. In this paper, we test several predictions based on the hypothetical scheme shown in Fig. 1. In particular, we manipulate alternate pathways in various pdx insertion mutants to gain new information about the pathways leading to pyridoxine. We also reexamine the possible roles of several putative intermediates, such as glycolaldehyde, ,-hydroxypyruvate, and D-alanine, in pyridoxine biosynthesis. The results are consistent with the pathway in Fig. 1, which in turn is consistent with all previous isotope labeling data. MATERIALS AND METHODS Materials. Enzymes used in cloning serC were the following: restriction endonucleases, T4 DNA ligase, and phosphorylated Sall linkers (New England BioLabs, Beverly, Mass.); T4 DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.); and bacterial alkaline phos-
* Corresponding author. 6518
PYRIDOXINE BIOSYNTHETIC PATHWAY IN E. COLI
VOL. 172, 1990
O 1NH HO-8-P-H
6519
GLYCINE
0 HOH
HM*HGLYCOLALDEHYDE
(ALTERNATE PATHWAY) \ 01 1 01 1 OII 0II s1 D-1-DEOXYC,-H NAD NADH C-OH NAD+ NADH C-OH Glu aKG C,-OH ( C-OH XYLULOSE C-H H-9-OH oil H-C,=O H-C,-NH HCH-C-NH H-C-OH H-C-OH H,C-OH D H-C-OH H-CSerC H9O®E4PD H-C- O-® Sr (?) H!C'O H!C'-OH C, H H ERYTHROSEERYTHRONATE39 I-HYDROXY4r--PHOSPHO4,-HYDROXY4-PHOSPHATE 4-PHOSPHATE 4t-PHOSPHOH YDROXYTHREONINE HIYDROXYT HREONINE at-KETOBUTYRATE
if'
H-9--®I
-
AC0A
A
PYRIDOXINE (VITAMIN BO)
0 NAD + NADH Glu a KG It C-OH \ , C-OH \ S C-OH ,C--OH H-C-OH - H-C-NH C o SerC H-C-Nra TO PROTEINS. H-CH-C -O-®i SerA H-¢C_o-(i) Sor H-C -OH AMINO ACIMS. TO AROMATIC H H H H PHOSPHOLIPIDS. AINO ACIDS 3-PHOSPHO3I-PHOSPHOa3-PHOSPHOENTEROCHELIN SERINE HIYDROXYGLYCERATE E51lIELNI!; F 'YRUVATE FIG. 1. Proposed pathway for the entrance of nitrogen 1 and carbons 5, 5', and 6 into the intermediate, 4-hydroxythreonine, and the pyridine ring of pyridoxine (vitamin B6). The major phosphorylated pathway of serine biosynthesis is drawn below and in parallel to the proposed branch of the pyridoxine pathway. An alternate pathway from glycine and glycolaldehyde that can be detected in pdxB and serC mutants is also shown (see text). The numbers on the nitrogen and carbon atoms refer to final positions of the pyridoxine ring, and the dot marks the carbon of 4-hydroxythreonine lost during decarboxylation. The pathway is based on genetic, physiological, and isotope labeling data (see text), and the intermediates shown have not yet been detected chemically. Enzyme designations: E4PD, erythrose-4-phosphate dehydrogenase; PdxB, likely erythronate-4-phosphate dehydrogenase (see text); SerC, phosphoserine aminotransferase; (?), hypothetical phosphatase activity encoded by pdxJ or another gene (see text); SerA, D-3-phosphoglycerate dehydrogenase; SerB, 3-phosphoserine v
1R
0-0)
phosphatase.
phatase (Bethesda Research Laboratories, Gaithersburg, Md.). Antibiotics, biochemicals, and crystalline D-erythrose-4-phosphate were purchased from Sigma Chemical Co. (St. Louis, Mo.). Ingredients for culture media were from Difco Laboratories (Detroit, Mich.). Inorganic salts and electrophoresis-grade agarose were bought from Fisher Scientific (Fair Lawn, N.J.). Bacterial strains, plasmids, media, and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Markers were moved between strains by generalized transduction using P1 kc or P1 vir bacteriophage as described previously (35). Recombinant plasmids were constructed using standard techniques (3, 41). Insertional mutagenesis of plasmids with mini-Mu dI(Km) and mini-Mu dII(KmD) was performed as previously described (10). The mini-Mu dl elements used (Table 1) form stable insertions that cannot transpose further (10). Insertion positions in plasmids were determined by restriction analysis using several enzymes (3, 41) and are accurate to within about 50 bp. Insertions imparting resistance to kanamycin were crossed from linearized plasmids into the chromosome of recBC sbc mutant JC7623 as detailed before (2). Chromosomal insertions were moved into Alac Atrp mutant VJS433 and Alac mutant NU816 to form isogenic sets of strains (Table 1). LBC rich medium was Luria-Bertani broth supplemented with 30 jig of L-cysteine per ml. Minimal Vogel-Bonner 1XE medium containing 0.01 mM FeSO4 was prepared as previously described (12). Supplements are listed in the table footnotes and figure legends. FeSO4 was omitted from
minimal salts medium containing the six aromatic compounds, L-Trp, L-Tyr, L-Phe, 4-aminobenzoate, 4-hydroxybenzoate, and 2,3-dihydroxybenzoate (see Table 4). For strains with complicated genotypes (Table 1), additional nutritional requirements were added at concentrations suggested by Davis et al. (12). Liquid cultures were grown with vigorous shaking in Nephelo flasks at the temperatures indicated, and their turbidities were monitored by using a Klett-Summerson colorimeter. Growth tests were performed by streaking cells onto prewarmed plates, by inoculating liquid cultures with a small amount of washed cells, or by using crystal spot tests (1). Pseudorevertants of pdxB::Kmr mutant NU402 that grew on minimal salts medium plus 0.4% glucose (MMG) at 37°C were selected as follows. A 10-ml LBC culture of NU402 was grown at 37°C to mid-exponential phase, collected by centrifugation, suspended in 10 ml of MMG, centrifuged, and washed one more time. Washed cells were suspended in 10 ml of MMG, and 0.1 ml of cells was spread onto prewarmed MMG or MMG plus 0.5% (wt/vol) ACH (vitamin assay acid casein hydrolysate) plates, which were incubated at 37°C for 3.5 days. Pseudorevertants were restreaked onto the same selective medium at 37°C, and single colonies were saved by patching them onto LBC plates containing 50 jig of kanamycin per ml. Properties of two of three groups of pdxB::Kmr pseudorevertants were stable to repeated streaking on nonselective LBC medium plus 50 jig of kanamycin per ml at 37°C (see Table 3). Pseudorevertants of serC:: mini-Mu d mutants NU1107 and NU1113 could also be
6520
LAM AND WINKLER
J. BACTERIOL.
TABLE 1. Bacterial strains and plasmids Strain or plasmid
Genotypea
Source or reference
E. coli K-12
CAG12168 CGSC2829 (formerly AB2829) CGSC4539 (formerly AT3143) CGSC4297 (formerly KL282) GS415 JC7623 NU402 NU426b NU816b NU934 NU979 NU988 NU989 NU1054C NU1055C NU1107 NU1113 NU1331C
NU1333 POI1734 (also called P01683) P0111734D
TT9813 VJS433 E. coli B or E. coli K-12/B WG5 (E. coli B)
WG532 (E. coli K-12/B) Plasmids pBR322 pGT-17 pNU169 pNU174C pNU178C pNU193C pNU194C pNU195C pSAWT
MG1655 zgd-210::TnlO (=30% cotransducible with serA) aroA354 supE42 X+
C. Gross (45) B. Bachmann collection
pdxC3 cycAl cycB2 his-53 ilv-277 lacY29 met-65 proC24 purE41 pyrF30 rpsL97 tonA32 tsx63 xylJ4 XA serCl3 ompF627 pitlO phoA4(Am) relAl (serSJ4serSI6) spoTi supD32 tonA32 T2r serA25 zgb224::TnlO supE44 gal-6 lacYl malAl mtl-2 thi-J tonA2 xyl-7 Xr/recB21 recC22 sbcl5 ara arg his leu pro thr W3110 pdxB: :
