INTRODUCTION

Our early work on the biosynthesis of vitamin B₆ in Escherichia coli mutants WG2 and WG3, based on studies originally using substrates labeled with ¹⁴C and ³H (1, 2, 3, 4), and later substrates singly labeled with ¹³C (5), established the pattern of incorporation into the skeleton of pyridoxol (1; see Structure 1) of the carbon atoms of glycerol, glucose, and pyruvic acid. From these results it was inferred that pyridoxol is constructed from three glucose-derived triose phosphates, two of which enter intact, supplying the C₃ fragments, C-3,4,4 and C-6,5,5 of pyridoxol, with the carbon atom holding the phosphate ester group yielding C-4 and C-5, while the third triose phosphate unit yields a C₂ unit, by decarboxylation of pyruvate, whose CH₃-CO moiety then supplies the C₂ unit, C-2,2, i.e. the CH₃ group and the adjacent ring carbon of pyridoxol.

The synthesis of potassium [2,3-¹³C₂]D-erythroate () from [1,2-¹³C₂]acetylene ().

We now furnish definitive proof that these inferences were indeed correct. This direct evidence comes from the results of incorporation studies with substrates labeled with ¹³C at contiguous sites, so called "bond-labeled" compounds, whose mode of incorporation into pyridoxol was determined by ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR).

The power of this method lies in the circumstance that, when contiguous carbon atoms within a compound are 100% enriched in ¹³C, this fact is indicated by the presence, within the ¹³C NMR spectrum of the compound, of ¹³C-¹³C coupling, which is indicated by the appearance of peaks that are not present in the spectrum of a natural abundance sample or of a singly ¹³C-enriched sample. These new peaks are detectable even if the fully ¹³C enriched sample is a minor component of a mixture that consists mainly of the unenriched compound.

The appearance of peaks due to ¹³C-¹³C coupling, in the signals of the ¹³C NMR spectrum of a biosynthetic product, as a result of incorporation of contiguously 100% ¹³C-¹³C-enriched (i.e. bond-labeled) substrates, provides direct evidence for the transfer from substrate into the biosynthetic product of an intact C-C unit. The application of bond-labeled samples thus constitutes a powerful tool for the demonstration of the transfer of intact multi-carbon fragments in biosynthetic investigations; provided adequate incorporation can be achieved in a tracer experiment with a ¹³C bond-labeled substrate, these are the tracers of choice. Neither radioactive tracer methods nor the use of substrates that are enriched with stable isotopes at single sites can show incorporation of intact multi-carbon units. Furthermore, ¹³C NMR does not only detect the site of labeling, but at the same time confirms the identity of the labeled sample and determines its degree of chemical purity. A precondition of the application of NMR methods for the analysis of biosynthetic incorporation patterns is the reliable assignment of each spectral signal to the individual atom that gives rise to it.

Support for the inferences drawn from our tracer studies with ¹⁴C-labeled substrates, that the pyridoxine skeleton was constructed from three glucose-derived subunits, came from an experiment with [1,2,3,4,5,6-¹³C₆]D-glucose (referred to as Experiment 1 in Table I) (6, 7), which demonstrated that, as predicted, only two carbon-carbon bonds, those between C-2 and C-3 and between C-4 and C-5, are newly formed in the course of the biosynthetic derivation of pyridoxol from glucose, and that glucose does indeed supply three intact multicarbon units, as the building blocks of the three fragments, C-2,2, C-3,4,4, and C-6,5,5, of pyridoxol (Fig. 1, A).

Table I. Experimental details Each experiment (except Experiment 9) consisted of five 1-liter incubations. Pyridoxol hydrochloride was isolated from the culture fluid of each of the five incubations, after removal of the cells by centrifugation and addition of unlabeled pyridoxol hydrochloride (2.5 mg) as carrier. Exp. no. Substrates Weight 13C NMR spectrum of isolated pyridoxol HCl mg/liter (mmol) 1 [1,2,3,4,5,6-13C6]-D-Glucose  200   (5.6) A (Fig. 1; Refs. 6 and 7) D-Glucose  800 2 [1,2-13C2]-D-Glucose  300   (5.6) B (Fig. 2) D-Glucose  700 3 Sodium [2,3-13C2]Pyruvate  150   (1.3) C (Fig. 2) D-Glucose 1000   (5.6) 4 Sodium [2,3-13C2]Pyruvate  200   (1.8) Not shown D-Xylosea  500   (3.3) (similar to C) 5 [1,2,3,4,5,6-13C6]-D-Glucose  200   (5.6) D (Fig. 3) D-Glucose  800 1-Deoxy-D-xylulose  750   (5.6) 6 [2,3-13C2]-1-Deoxy-D-xylulose  200   (1.5) F (Fig. 4) D-Xylosea  500 (3.3) 4-Hydroxy-L-threonineb  100   (0.74) L-Threoninec   20   (0.17) 7 [1,2,3,4,5,6-13C6]-D-Glucose  200   (5.6) E (Fig. 3) D-Glucose  800 4-Hydroxy-L-threonine  750   (5.5) L-Threoninec   20   (0.17) 8 [2,3-13C2]-4-Hydroxy-L-threonine  160   (1.2) G (Fig. 5) D-Glucose  500   (2.8) L-Threoninec   20   (0.17) 9d Potassium [2,3-13C2]-D-erythroate  200   (1.1) H (Fig. 5) D-Xylosea  500   (3.3) a  In Experiments 4, 6, and 9, D-xylose in place of D-glucose was used as the general carbon source in order to avert the possibility that the presence of glucose might limit the uptake and incorporation of the labeled carbohydrate substrates, [2,3-13C2]-1-deoxy-D-xylulose (Experiment 6) and [2,3-13C2]-D-erythroate (Experiment 9). Experiment 4 served as a test of D-xylose as the general carbon source. This change in the general carbon source was made after consideration of the results of the D-glucose displacement experiments, Experiments 5 and 7, and after failure to observe 13C incorporation in an early experiment with [2,3-13C2]-1-deoxy-D-xylulose. Whereas unlabeled 4-hydroxy-L-threonine completely suppressed the incorporation, into C-6,5,5 of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 7), unlabled 1-deoxy-D-xylulose only partially suppressed the incorporation, into C-2,2,3,4,4 of pyridoxol, of 13C from [1,2,3,4,5,6-13C6]-D-glucose (Experiment 5). Furthermore, in an experiment with [2,3-13C2]-1-deoxy-D-xylulose in which D-glucose served as the general carbon source, no 13C enrichment was detectable in the pyridoxol that was isolated. We reasoned that these results may have been the consequence of an "inducer exclusion effect," a phenomenon that occurs in bacterial systems, whereby certain carbohydrates (so-called "PTS-carbohydrates"), e.g. glucose, inhibit the transport and metabolism of other carbohydrates (so-called "class I non-PTS carbohydrates") (PTS = phosphoenolpyruvate:carbohydrate phosphotransferase system) (16) of which 1-deoxy-D-xululose might be one. We surmised that in our short term (6 h) incubations, D-glucose might have inhibited the uptake of 1-deoxy-D-xululose, whereas entry of the amino acid, 4-hydroxy-L-threonine, had not been affected. If this reasoning were correct, the problem might be overcome by using as the general carbon source in this experiment a non-PTS-carbohydrate such as D-xylose, in place of D-glucose, a PTS-carbohydrate. Cultures of E. coli mutant WG2 were established on the minimal medium, with D-xylose as the general carbon source, and in a test experiment (Experiment 4) it was found that under these conditions incorporation of label from sodium [2,3-13C2]pyruvate matched the result obtained when D-glucose served as the general carbon source (Experiment 3). Having thereby established that D-xylose could replace D-glucose as the general carbon source without measurable impairment of pyridoxine biosynthesis, we proceeded with Experiments 6 and 9 (Table I). b  When this experiment (Experiment 6) was performed, it was already known, from Experiments 7 and 8, that 4-hydroxy-L-threonine serves as a direct precursor of pyridoxol. A sample of the amino acid was added since it was observed that this stimulates pyridoxol biosynthesis. c  4-Hydroxy-L-threonine shows antimetabolite properties in E. coli, inhibiting the growth of E. coli B when cultured on a pyridoxine-supplemented growth medium. Addition of L-threonine (20 mg/liter) permits the mutant to grow in the presence of the amounts of 4-hydroxy-L-threonine that were added to the medium in Experiments 6, 7, and 8 (100, 750, and 160 mg/liter, respectively). d  Only three 1 liter cultures (rather than five) were employed in this experiment.

The results of the tracer studies with ¹³C bond-labeled substrates that are here presented define more precisely the mode of incorporation of glucose carbon atoms into the glucose-derived subunits, C-2,2, C-3,4,4 and C-6,5,5, of pyridoxol. They show, further, that an intact C₂ unit, derived from C-3,2 of pyruvic acid, serves as the precursor of the C₂-unit, C-2,2, and that the C₅ chain, C-2,2,3,4,4 of pyridoxol, originates by linear combination of the pyruvate-derived C₂ unit and one of the glucose-derived C₃ units. Furthermore, the results establish the identities of the two multicarbon precursors whose union accounts for the formation of the complete skeleton of pyridoxine. It is shown that 1-deoxy-D-xylulose (12) supplies the intact C₅ chain, C-2,2,3,4,4, while the C₃N unit, N-1,C-6,5,5, is derived intact from 4-hydroxy-L-threonine (13), which is thereby proven to be an intermediate of the biosynthetic route. D-Erythroate (11) is shown to serve as an intermediate between glucose and 4-hydroxy-L-threonine. Preliminary reports of part of this work have appeared elsewhere (8, 9, 10).

EXPERIMENTAL PROCEDURES

Organism

The organism used in these investigations was E. coli B strain WG2. This is a pyridoxine auxotroph (pdxH) that lacks pyridoxol-phosphate oxidase (EC 1.1.1.65 or EC 1.4.3.65).

Media

This was a nutrient broth medium (Oxoid Ltd., London, United Kingdom) prepared according to the supplier's instructions.

The minimal salts medium contained the following salts: 7 g/liter KH₂PO₄, 3 g/liter K₂HPO₄, 1 g/liter (NH₄)₂SO₄, 0.1 g/liter MgSO₄, and 0.01 g/liter CaCl₂. D-Glucose (Experiments 1-3, 5, 7, and 8) or D-xylose (Experiments 4, 6, and 9) served as the general carbon source. Pyridoxal hydrochloride was added to a concentration of 6 × 10⁷ M when the minimal medium was used to grow the pdxH mutant.

All media were prepared in distilled water and were sterilized by autoclaving. The pyridoxal hydrochloride supplement solution was sterilized by filtration.

Stock Cultures

Stock cultures of E. coli B WG2 were maintained on monthly slants of the nutrient broth medium. After subculturing from the previous month's stock, fresh slants were incubated 24 h at 37 °C and were then stored at 4 °C. Every time fresh stock slants were prepared, slants of minimal salts medium, with and without pyridoxal supplementation, were inoculated and incubated 24 h at 37 °C in order to monitor for the presence of wild-type revertants.

Labeled Compounds

The following labeled compounds were acquired from a commercial source (Cambridge Isotope Laboratories, Inc. (CIL)): [1,2,3,4,5,6-¹³C₆]D-glucose (98% ¹³C), [1,2-¹³C₂]D-glucose (99% ¹³C), and sodium [2,3-¹³C₂]pyruvate (99% ¹³C).

The following labeled compounds were prepared from commercial starting materials by multi-step syntheses devised for the purpose. [2,3-¹³C₂]4-Hydroxy-L-threonine was synthesized (11) from [1,2-¹³C₂]acetylene (99% ¹³C) (CIL) in eight steps with an overall yield of 13%. [2,3-¹³C₂]1-Deoxy-D-xylulose was synthesized (12) from ethyl bromo[1,2-¹³C₂]acetate (99% ¹³C) (CIL) in 14 steps with an overall yield of 15%. Potassium [2,3-¹³C₂]D-erythroate (11) was synthesized (Scheme 1) from [1,2-¹³C₂]acetylene (99% ¹³C) (2) (CIL) in nine steps with an overall yield of 20%.

The synthesis of potassium [2,3-¹³C₂]D-erythroate () from [1,2-¹³C₂]acetylene ().

Molecules that are fully enriched with ¹³C at contiguous sites, particularly if such molecules have C_S symmetry, yield strongly coupled ¹³C NMR spectra. The enriched carbon atoms and their neighbors form a "deceptively simple" ABX system (13). The coupling constants were determined by simulating the spectra using the XSIM computer program of Dr. Kirk Marat, Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.

[2,3-¹³C₂]-(Z)-But-2-ene-1,4-diol (4) (1.05 g, 11.6 mmol) (prepared in two steps from [1,2-¹³C₂]acetylene (2) (CIL) via [2,3-¹³C₂]but-2-yne-1,4-diol (3); Ref. 11) was dissolved in dry N,N-dimethylformamide (35 ml) and imidazole (3.40 g, 50 mmol) was added. tert-Butyldiphenylsilyl chloride (6.5 ml, 25 mmol) was added dropwise by syringe, and the mixture was stirred 4 h at room temperature. It was then poured into diethyl ether (150 ml) and extracted first with water (150 ml), then with aqueous hydrochloric acid (~5%, 2 × 50 ml), with saturated sodium bicarbonate solution (50 ml) and again with water (50 ml). The organic layer was dried (MgSO₄), the solvent evaporated, and the residue chromatographed on silica gel (petroleum ether, followed by diethyl ether/petroleum ether 1:5 v/v), to yield the product (5) as a colorless oil (6.01 g, 91.6%).

¹H NMR (200 MHz, CDCl₃):  7.56-7.61 (m, 8H), 7.23-7.73 (m, 12H), 5.06-6.22 (dm, ¹J_C,H = 156 Hz, 2H), 4.07 (d, ³J_{H, H} = 3.74 Hz, 4H), 0.97 (s, 18H).

¹³C NMR (75.5 MHz, CDCl₃):  135.5, 133.6, 129.9 (enriched), 128.8, 127.6, 60.5 (X part of a deceptively simple ABX system, ¹J_AB = 70 Hz, ¹J_AX = 45 Hz, ²J_BX = 3 Hz), 26.8, 19.1.

IR¹ (film): 1111 (v_max) cm¹.

ms (CI): m/z 584 (30%, m + NH₄⁺), 311 (100%).

[2,3-¹³C₂]1,4-Di(tert-butyldiphenylsilyloxy)-(Z)-but-2-ene (5) (6.01 g, 10.6 mmol) was dissolved in tert-butanol (21 ml). Tetrahydrofuran (8 ml), water (4 ml), and N-methylmorpholine N-oxide (1.60 g, 13.7 mmol) were added, and the air above the mixture was displaced by nitrogen. An aqueous solution of osmium tetroxide (4% w/v, 4 ml) was added and the mixture stirred 4 h at room temperature, after which time no starting material remained (TLC: Silica gel 60, ethyl acetate/hexane 1:4). Sodium bisulfite (125 mg) in water (5 ml) was added, followed by Florisil^® (5.25 g) and the mixture was stirred 10 min and was then filtered through silica gel and eluted with ethyl acetate (250 ml). Solvent was evaporated and the residue recrystallized from hexane (30 ml), yielding colorless crystals of the product (6), m.p. 90 °C (3.48 g, 54.6%). The mother liquor was concentrated and the residue chromatographed (Silica gel 60, diethyl ether/petroleum ether 1:7, v/v), yielding a further 1.11 g (17.4%) of product, together with a mixed fraction from which an additional 189 mg of 6 was obtained by crystallization. Total yield was 4.78 g (75.0%).

¹H NMR (300 MHz, CDCl₃):  7.69-7.73 (m, 8H), 7.26-7.50 (m, 12H), 3.50-4.13 (dm, ¹J_{C, H} = 141 Hz, 2H), 3.92 (s, 4H), 2.75 (s (br.) 2H), 1.11 (s, 18H).

¹³C NMR (75.5 MHz, CDCl₃):  135.5, 133.0, 129.8, 127.8, 71.9 (enriched), 65.1 (X part of a deceptively simple ABX system, ¹J_AB = 43 Hz, ¹J_AX = 36 Hz, ²J_BX = 6 Hz), 26.9, 19.2.

IR (KBr): 3511, 692 (v_max) cm¹.

ms (CI): m/z 618 (15%, m + NH₄⁺), 367 (100%), 131 (90%).

Diol (6) (4.78 g, 7.95 mmol) was dissolved in dry benzene (100 ml). 2,2-Dimethoxypropane (15 ml, 122 mmol) was added and the mixture heated to 80 °C. Pyridinium p-toluenesulfonate (275 mg) was added and methanol removed by azeotropic distillation (b.p. 54 °C), until the reaction was complete (~1 h). Benzene (~75 ml) was distilled off, the mixture cooled to room temperature, and diethyl ether added. The solution was washed with water (2 × 50 ml), dried (MgSO₄), and concentrated in vacuo to yield the dioxolane (7) (5.05 g, 100%).

¹H NMR (200 MHz, CDCl₃):  7.57-7.64 (m, 8H), 7.24-7.39 (m, 12H), 4.45-4.75 (m, 1H), 3.62-4.00 (m, 5H), 1.36 (s 3H), 1.32 (s, 3H), 0.96 (s, 18H).

¹³C NMR (75.5 MHz, CD₂Cl₂):  135.6, 135.5, 133.5, 133.4, 129.6, 127.7, 108.4, 77.4 (enriched), 63.2 (X part of a deceptively simple ABX system, ¹J_AB = 37 Hz, ¹J_AX = 40 Hz, ²J_BX = 5 Hz), 27.8, 26.7, 25.3, 19.1. 

IR (film): 1112 (v_max) cm¹.

ms (CI): m/z 658 (25%, m + NH₄⁺), 565 (95%), 385 (100%).

Disilyl ether (7) (5.05 g, 7.95 mmol) was dissolved in tetrahydrofuran (40 ml). Water (0.5 ml, 28 mmol) and a solution of tetra-n-butylammonium fluoride in tetrahydrofuran (1 M, 24 ml) were added and the mixture kept 90 min at room temperature. Florisil^® (8.5 g) was then added with stirring, which was continued for 10 min, and the mixture was then filtered through a pad of silica gel and eluted with ethyl acetate/methanol (1:1 v/v, 100 ml). The residue, obtained after evaporation of the solvent, was chromatographed on silica gel (ethyl acetate/hexane, 1:2 v/v, followed by ethyl acetate/methanol 1:1 v/v). The oily product was further purified by vacuum distillation (Kugelrohr, 1 mm Torr, 145 °C). Diol (8) was obtained as an oil (1.20 g, 93%), which crystallized (m.p. 47 °C; literature m.p. of the unenriched compound 38 °C (Ref. 14), 46-47 °C (Ref. 15)) after 2 weeks in the refrigerator.

¹H NMR (200 MHz, CDCl₃):  4.27 (dm, ¹J_C,H = 146 Hz, 2H), 3.77 (s (br), 4H), 2.51 (s (br), 2H), 1.45 (s, 3H), 1.35 (s, 3H).

¹³C NMR (75.5 MHz, CDCl₃):  108.4, 76.8 (enriched), 60.8 (X part of a deceptively simple ABX system, ¹J_AB = 38 Hz, ¹J_AX = 41 Hz, ²J_BX = 4 Hz), 27.6, 25.1.

IR (KBr): 3300, 1041.2 (v_max) cm¹.

ms (CI): m/z 165 (100%, m + 1⁺).

Diol (8) (1.20 g, 7.3 mmol) was dissolved in tert-butyl methyl ether (20 ml), vinyl acetate (1 ml, 10.8 mmol) was added, followed by a sample of lipase from Pseudomonas fluorescens (SAM 2, 80 mg, 3360 units), and the mixture was stirred 4 d at 35 °C. The solid was then filtered off and washed with tert-butyl methyl ether (80 ml). The filtrate was evaporated in vacuo and the residue chromatographed on silica gel (diethyl ether) to yield enantiomerically pure monoacetate (9) (1.11 g, 74%). []_D²⁵ = +17.9° (c = 1.00, CHCl₃) (literature []_D of the unenriched compound []_D²⁰ = +16.6° (c = 1, CHCl₃) (14).)

¹H NMR (200 MHz, CDCl₃):  4.53-4.72 (m, 1H), 4.17-4.26 (m, 1H), 3.80-4.10 (m, 2H), 3.36 (s (br.) 2H), 2.49 (s (br.), 1H), 2.01 (s, 3H), 1.41 (s, 3H), 1.30 (s, 3H).

¹³C NMR (50.3 MHz, CDCl₃):  171.0, 108.9, 77.9 (enriched, d_AB, ¹J_{C, C} = 34 Hz), 74.5 (enriched, d_AB, ¹J_C,C = 34 Hz), 63.3 (d, ¹J_C,C = 44 Hz), 61.2 (d, ¹J_C,C = 41 Hz), 27.5, 25.0, 20.7.

IR (film): 3468, 1742, 1044 (v_max) cm¹.

ms (CI): m/z 224 (90%, m + NH₄⁺), 207 (100% m + 1⁺).

Alcohol (9) (0.36 g, 1.7 mmol) was dissolved in acetonitrile (3.5 ml). Carbon tetrachloride (3.5 ml), water (5.5 ml), and sodium periodate (1.50 g, 70.2 mmol) were added and air above the mixture was displaced by nitrogen. Ruthenium trichloride trihydrate (50 mg, 0.2 mmol) was added and the mixture stirred vigorously 3 h at room temperature. Brine (50 ml) and methylene chloride (50 ml) were added, and the aqueous layer was extracted with methylene chloride (4 × 50 ml). The combined organic phase was dried (MgSO₄) and the solvent removed in vacuo. The residue was taken up in diethyl ether (5 ml) and filtered through Celite^®. Evaporation of the solvent gave the product (10) as a colorless oil (364 mg, 94%). []_D²⁰ = +18.5° (c = 1.01, CHCl₃).

¹H NMR (200 MHz, CDCl₃):  8.8-9.5 (s(br.), 1H), 4.97-5.12 (m, 1H), 4.08-4.51 (m, 3H), 2.08 (s, 3H), 1.61 (s, 3H), 1.42 (s, 3H).

¹³C NMR (50.3 MHz, CDCl₃):  172.9, 170.4 (d, ¹J_C,C = 51 Hz), 111.5, 75.3 (enriched, d_AB, ¹J_C,C = 33 Hz), 75.0 (enriched, d_AB, ¹J_C,C = 33 Hz), 62.5 (d, ¹J_C,C = 42 Hz), 26.7, 25.0, 20.6.

IR (film): 3401 (v_max), 1771, 1729 cm¹.

ms (CI): m/z 238 (M + NH₄⁺, 5%), 198 (100%).

Acid (10) (222 mg, 1.0 mmol) was dissolved in acetonitrile (7 ml). A 5:1 mixture of water and concentrated hydrochloric acid (3 ml) was added, and the mixture was stirred 3 h at room temperature. The solution was concentrated in vacuo, the residue dissolved in methanol (2 ml), a methanolic solution of potassium hydroxide (2.5 M, 1 ml) was added, and the mixture stirred 90 min at room temperature. Addition of diethyl ether (5 ml) induced the product to crystallize. The crystalline product was filtered off, washed with diethyl ether (10 ml), and dried, yielding crude potassium erythroate (174 mg) as a yellow powder.

The crude product (from several runs) (855 mg) was dissolved in water (1.7 ml), methanol (5.1 ml) was added, and the mixture was stored 2 d in the refrigerator. Pure potassium erythroate (11) (650 mg, 76%) was obtained as colorless leaflets. []_D²⁰ = +11.0° (c = 1.12, H₂O).

¹H NMR (200 MHz, D₂O):  4.14-4.43 (m, 1H), 3.24-3.62 (m, 3H).

¹³C NMR (50.3 MHz, D₂O):  178.5 (X part of a deceptively simple ABX system, _AB = 28 Hz, ¹J_AB = 42 Hz, ¹J_AX = 70 Hz, ²J_BX = 8 Hz), 74.0 (enriched, d_AB, ¹J_CC = 40 Hz), 73.1 (enriched, d_AB, ¹J_CC = 40 Hz), 61.9 (X part of a deceptively simple ABX system (_AB = 13 Hz, ¹J_AB = 40 Hz, ¹J_AX = 45 Hz, ²J_BX = 3 Hz).

IR (KBr): 3405, 3194, 1717, 1605 (v_max) cm¹.

ms (ES): m/z 137 (100% m⁺).

Incubation in the Presence of Labeled Compounds

Each tracer experiment was started by subculturing from the current month's nutrient broth stock slant onto a pyridoxal-supplemented minimal medium slant, which had been prepared with either 0.5% w/v D-glucose (Experiments 1-3, 5, 7, and 8) or 0.5% w/v D-xylose (Experiments 4, 6, and 9) as the carbon source. The slant was incubated 24 h at 37 °C. Cells from this slant were then used to inoculate 2 × 500-ml samples of the same medium, one of which was supplemented with pyridoxal hydrochloride, the other without pyridoxal supplementation. These cultures were incubated on a rotary shaker (New Brunswick Scientific) at 37 °C, until an optical density measurement at 600 nm indicated that growth in the supplemented culture was well into the exponential phase (approximately 12 h). Any growth in the unsupplemented culture served to indicate the presence of wild-type revertants or possible contaminants. If this were observed, the experiment could have been aborted at this stage without any wastage of labeled substrate. Fortunately, in our hands the pdxH mutation in strain WG2 is very stable, and so far such an occasion has not arisen. Nevertheless, the control unsupplemented culture was included in every experiment as a precautionary measure.

The cells from the pyridoxal-supplemented culture were harvested by centrifugation (10 min at 7000 rpm) and washed with sterile 0.9% saline (3 × 100 ml). The washed cells were divided into two equal portions, each one of which was resuspended in minimal salts medium (500 ml) without pyridoxal but containing the appropriate carbon source, the labeled substrate and other addends. These cultures were incubated 6 h on the rotary shaker (400 rpm, 37 °C). In order to obtain sufficient material for NMR investigation, each incubation was repeated five times, except in Experiment 9, when labeled substrate for only three repeat incubations was available. Details of the components used in each of the experiments are presented in Table I.

Isolation of Pyridoxol from the Culture Medium

The contents of each 500-ml culture flask from each of the two 500-ml incubation experiments with ¹³C-labeled substrate were centrifuged 10 min at 7000 rpm, and the supernatant solutions decanted and combined. The solution was concentrated to 200 ml in vacuo on a rotary evaporator. The concentrated solution was treated with acid to hydrolyze phosphate esters; sulfuric acid (1 M, ~80 ml) was added until the final pH of the solution was 1.5, and the mixture was autoclaved (121 °C, 3 h). The solution was then evaporated to dryness in vacuo on a rotary evaporator. The residue was dissolved in potassium acetate/acetic acid buffer (0.2 M, pH 4.5, 200 ml) and the solution was filtered through a fine sintered glass filter. Pyridoxol hydrochloride (2.5 mg) was added to the filtrate as a carrier. This solution was then subjected to ion exchange chromatography, as follows.

A cation exchange column was prepared as follows. Dowex 50-X8 (200-400 mesh) was washed, in succession, with water, hydrochloric acid (3 M), water, potassium hydroxide solution (6 M), and water, until free of fine particles. This material was loaded into a column (15 × 1.5 cm), which was then washed with potassium hydroxide (6 M), followed by water, until the pH of the eluate was approximately pH 9.

The solution containing the concentrate of the incubation (pH 4.5, 200 ml, see above) was applied to this column. Elution of the column was carried out by stepwise increase in pH, with the following buffer sequence: potassium acetate (0.2 M)/acetic acid (0.2 M), pH 5.0, 100 ml; potassium acetate (0.2 M)/acetic acid (0.2 M), pH 5.5, 100 ml; potassium acetate (0.2 M)/acetic acid (0.2 M), pH 6.0, 50 ml; boric acid (0.2 M)/ potassium chloride (0.2 M)/acetic acid (0.2 M), 200 ml, adjusted to pH 6.6 by addition of sodium hydroxide (0.2 M). Fractions (10 ml) were collected and assayed by UV spectroscopy in order to determine the position of pyridoxol in the elution sequence. Pyridoxol was eluted in the first six 10-ml fractions following the addition of the final buffer (pH 6.6).

A second Dowex 50-X8 (200-400-mesh) column (6 × 1 cm) was prepared and washed with hydrochloric acid (0.1 M) until the eluate was acidic. The pyridoxol fractions from the first column were pooled, evaporated under reduced pressure, and redissolved in hydrochloric acid (0.1 M, 50 ml). The solution was then applied to the second column, which was eluted with distilled water until the eluate was neutral, and then with dilute ammonia (3%).

Up to this stage, each 1-liter incubation from each of the experiments was worked up separately. Now, the ammoniacal eluates (10 ml) from the repeat incubations (3 × 1 liter in Experiment 9, 5 × 1 liter in all other experiments) were pooled. The pooled solution was evaporated under reduced pressure and the solid residue dissolved in a small volume of anhydrous methanol (2 ml). This solution was applied to a preparative plate for thin layer chromatography (thickness 2 mm, Silica Gel G according to Stahl). Development was for 15 cm in the solvent system tert-butyl alcohol/methylethylketone/ammonia (0.880)/water (4:3:2:1). The plate was dried, and the pyridoxol band was identified by its characteristic blue fluorescence under UV light (254 nm). The band was removed from the plate, and the pyridoxol extracted from the silica gel by stirring overnight at 40 °C in anhydrous methanol (40 ml). The resultant slurry was filtered through a fine sintered glass filter. The filtrate was reduced to small volume (1-2 ml), and three drops of hydrochloric acid (0.1 M) were added. On addition of anhydrous ether, pyridoxol hydrochloride crystallized.

Final purification was effected by sublimation at 125-130 °C and 2 × 10³ mm. In most instances the yield of sublimed pyridoxol hydrochloride, m.p. 205-206 °C (with decomposition) (literature m.p. of the unenriched compound 204-205 °C (with decomposition ) (17); 206-208 °C (18)), was 8-9 mg, that is ~75-80% of the total weight of unenriched carrier that had been added. The sublimed product was dissolved in D₂O and the solution used for ¹³C NMR studies.

A summary of the ¹³C NMR parameters of pyridoxol hydrochloride is presented in Table II.

Table II. 13C NMR spectrum of pyridoxol (1); chemical shifts and coupling constants Carbon atom Chemical shift () 13C-13C coupling constants (± 0.5 Hz) C-2 16.5 1J2,2 46.4 C-4 59.0 1J4,4 45.1 C-5 60.1 1J5,5 48.0 C-6 131.8 2J5,6 3.7 C-5 138.8 1J5,6 64.9 C-4 142.5 C-2 144.7 1J2,3 73.0 C-3 154.7 1J3,4 64.5

RESULTS AND DISCUSSION

The ¹³C NMR spectrum (Fig. 1, A) of the sample of pyridoxol hydrochloride isolated from a culture of E. coli B mutant WG2 after incubation with [1,2,3,4,5,6-¹³C₆]D-glucose (6, 7) shows that the three fragments C-2,2, C-3,4,4 and C-6,5,5 are derived from glucose as intact multi-carbon units (Scheme 2, A). However, the spectrum does not provide information concerning the identity of the fragments of glucose from which these units are derived.

The labeling pattern of pyridoxol, derived from [1,2,3,4,5,6-¹³C₆]glucose, and from [1,2,3,4,5,6-¹³C₆]glucose in the presence of 1-deoxy-D-xylulose () or of 4-hydroxy-L-threonine ().

The answer to this question can now be conclusively deduced from three sets of labeling data. First, the ¹³C NMR spectrum (Fig. 2, B)² of the sample of pyridoxol from D-[1,2-¹³C₂]glucose (Experiment 2) shows that each of the signals due to the carbon atoms of the three carbon pairs C-2,2 ( 16.5, 144.7, ¹J_2,2 = 46.4 Hz), C-4,4 ( 59.0, 142.5, ¹J_4,4 = 45.1 Hz), and C-5,5 ( 60.1, 138.8, ¹J_5,5 = 48.0 Hz) appears as a "triplet," composed of a central natural abundance signal, originating from the carrier material that had been added to facilitate isolation, straddled by a doublet, due to ¹³C-¹³C enrichment at contiguous carbon atoms. No satellites appear at the signals due to C-3 ( 154.7) or C-6 ( 131.8). Thus, the spectrum shows that the labeled C₂ unit of the precursor had entered the three C₂ units C-2,2, C-4,4, and C-5,5 of pyridoxol intact. Second, it had been found earlier (3) that label from [1-¹⁴C]D-glucose (or from [6-¹⁴C]D-glucose) entered the three carbon atoms, C-2, C-4, and C-5 of pyridoxol, and only these three carbon atoms. This defines the orientation of entry into pyridoxol of the intact multicarbon units of glucose. The glucose carbon atoms yielding the three intact fragments derived from [1,2,3,4,5,6-¹³C₆]D-glucose enter as follows: C-1,2 (or C-6,5)³ of glucose yield carbon atoms C-2 and C-2, respectively, of the C₂ unit C-2,2 of pyridoxol; and carbon atoms C-1,2,3 (or C-6,5,4)³ of glucose enter C-4 and C-5, C-4 and C-5, and C-3 and C-6, respectively, of the two C₃ units C-4,4,3 and C-5,5,6 of pyridoxol. The early inference that the two C₃ units C-4,4,3 and C-5,5,6 of pyridoxol were derivable intact from C-1,2,3 (or C-6,5,4)³ of D-glucose, with C-1 (or C-6)³ of the sugar giving rise to C-2, C-4, and C-5, is thus confirmed, lending strong support to the early conclusion that the two C₃ units C-4,4,3 and C-5,5,6 of pyridoxol are derivable intact from triose phosphates.⁴

Third, it is now confirmed (Fig. 2, C) by the experiments with sodium [2,3-¹³C₂]pyruvate (Experiments 3 and 4) that the C₂ unit, C-2,2, of pyridoxol, inferred (3) to be derived intact from the CH₃-CO- fragment of pyruvic acid, is indeed so derived. Satellites appear only at the signals due to C-2 and C-2 ( 16.5, 144.7, ¹J_2,2 = 46.4 Hz).

The stage is thus set for an investigation of the identity of compounds that might serve as more advanced precursors of the B₆ skeleton. Since only two new C-C bonds, C-2,3 and C-4,5, are generated in the course of pyridoxol biosynthesis from D-glucose (Scheme 2, A) and since it may reasonably be assumed that these two bonds are not generated simultaneously, only two sets of advanced precursors need to be considered.

One alternative set of advanced precursors consists of a C₅ compound and a C₃N unit. The C₅ compound would be generated by closure of the bond destined to become the C-2,3 bond of pyridoxol, as the precursor of the C₅ unit, C-2-2-3-4-4 of pyridoxol, originating by union of the CH₃-CO fragment of pyruvic acid (C-2-2) with a triose phosphate (C-3-4-4). The remaining portion of the pyridoxol skeleton would then require a C₃ precursor of the fragment C-6-5-5, or possibly a C₃N precursor of the fragment N-1-C-6-5-5.

The other alternative assumes closure of the bond destined to become the C-4,5 bond of pyridoxol. The advanced precursors would then be a branched chain C₆ compound, serving as the source of the C₆ unit, C-3-4(-4)-5(-5)-6, while the remaining portion of the pyridoxol skeleton, C-2,2,N-1, would require a C₂N unit derived from the CH₃-CO fragment of pyruvic acid, together with a nitrogen source.

In the first instance we decided to focus on the former of the two alternatives, a possible origin of the pyridoxol skeleton by union of a C₅ plus C₃N unit. This turned out to be the right decision. This alternative demands that the C₅ unit must be derived from the CH₃-CO fragment of pyruvate plus a triose phosphate. The reaction of a triose phosphate, D-glyceraldehyde 3-phosphate (or of D-glyceraldehyde itself), with pyruvic acid, catalyzed by pyruvate dehydrogenase (EC 1.2.4.1), occurs in many micro-organisms, including E. coli (21, 22). The reaction is accompanied by loss of the pyruvic acid carboxyl group, to yield a C₅ compound, 1-deoxy-D-xylulose 5-phosphate (or 1-deoxy-D-xylulose (12, Scheme 2), respectively).

Preliminary evidence that 1-deoxy-D-xylulose serves as a precursor, supplying the C₅ chain, C-2,2,3,4,4 came from an experiment with [1,1,1-(RS)-5-²H₄]1-deoxy-D-xylulose. Deuterium from this substrate entered pyridoxol in the predicted manner (23). It remained to generate conclusive evidence that 1-deoxy-D-xylulose is indeed an intermediate on the route from glucose into pyridoxol and that it is incorporated intact. To prove intermediacy, it had to be demonstrated that the presence of excess 1-deoxy-D-xylulose in the system spared the incorporation of glucose carbon into the C₅ unit C-2,2,3,4,4 of pyridoxol. To prove intact incorporation, it had to be demonstrated that the bond, generated in the formation of the compound, i.e. the bond destined to become C-2,3 of pyridoxol, remains intact in the course of entry of 1-deoxy-D-xylulose into pyridoxol.

Evidence that each of the two conditions is fulfilled is provided by Experiments 5 and 6, respectively. The ¹³C NMR spectrum (Fig. 3, D) of the sample of pyridoxol (Scheme 2, D), isolated after administration of [1,2,3,4,5,6-¹³C₆]D-glucose in the presence of excess 1-deoxy-D-xylulose (12, Scheme 2), shows that the signals due to ¹³C enrichment at C-2 ( 16.5), C-2 ( 144.7), C-3 ( 154.7), C-4 ( 142.5) and C-4 ( 59.0) are less intense than the corresponding signals in the spectrum of pyridoxol derived from [1,2,3,4,5,6-¹³C₆]D-glucose alone (Fig. 1, A), whereas the signals due to C-6 ( 131.8), C-5 ( 138.8), and C-5 ( 60.1) remained unchanged (8). The extent of decrease in the incorporation of ¹³C into the carbon atoms C-2, C-2, C-3, C-4, and C-4, relative to that into carbon atoms C-6, C-5, and C-5, is illustrated in the expanded spectrum (Fig. 3, D), which shows that the satellites at the signal due to C-4 ( 59.0) are approximately 2.5 × less intense than those at the signal due to C-5 ( 60.1). Thus, the presence of excess 1-deoxy-D-xylulose had reduced the level of incorporation of glucose-derived ¹³C into the C₅ unit, C-2,2,3,4,4 of pyridoxol. It follows that 1-deoxy-D-xylulose lies on the route from glucose into pyridoxol. The ¹³C NMR spectrum (Fig. 4, F) of the sample of pyridoxol, isolated after administration of [2,3-¹³C₂]1-deoxy-D-xylulose (12) (Experiment 6) shows satellites at the signals due to C-2 and C-3 ( 144.7, 154.7, ¹J_2,3 = 73.0 Hz) and nowhere else, proving incorporation of the substrate without cleavage of its C-2,3 bond (10).

D

E

F

The evidence is thus complete that the intact C₅ chain of 1-deoxy-D-xylulose serves as source of the C₅ unit C-2,2,3,4,4 of pyridoxol. It remains for the future to determine whether it is the free deoxypentulose or its 5-phosphate ester that serves as the actual precursor.⁵

We now turn our attention to the identity of the C₃N precursor of N-1,C-6,5,5 of pyridoxol.

Since the C₃ unit C-6,5,5 is generated from C-3,2,1 (or from C-4,5,6) of glucose, we postulated (2) in our original working hypothesis that a triose phosphate is implicated in the derivation of this unit. Difficulties arose with this notion when it was discovered that the C₂ unit, C-5,5, was derivable from glycolaldehyde (25, 26) and that the CN unit, C-6,N-1, originated intact from the fragment -CH₂-NH₂ of glycine (27). On the basis of these results, we inferred (20) that the C₃N fragment, N-1,C-6,5,5, of pyridoxol might be derived from 4-hydroxy-L-threonine, which in turn might be formed by an aldolase type condensation of glycolaldehyde plus glycine, in the manner of the formation of L-serine from a formaldehyde equivalent plus glycine, catalyzed by serine hydroxymethylase (EC 2.1.2.1). In an attempt to achieve a reconciliation of the incorporation results with glucose on the one hand and with glycolaldehyde and glycine on the other, we postulated (20) intermediacy of 1-aminopropane-2,3-diol, which might arise either from 4-hydroxy-L-threonine by decarboxylation, or from D-glyceraldehyde by transamination. An attempt to support this notion experimentally failed. Label from a ²H-labeled sample of 1-aminopropane-2,3-diol was not incorporated into pyridoxol (28).

A solution to the problem was conceived by Lam and Winkler (29), who suggested, on the basis of genetic studies, that the glucose-derived C₃ unit, C-6,5,5, of pyridoxol was generated not by way of a glycolytic triose phosphate intermediate, but via intermediates originating from the pentose phosphate pathway. The major impetus for this idea was the parallelism, genetic, enzymic and structural, between the conversion of D-glyceraldehyde 3-phosphate to L-serine (via D-glyceric acid 3-phosphate, 3-hydroxypyruvic acid 3-phosphate, and 3-phosphoserine) and the conversion, in the homologous series, of D-erythrose 4-phosphate into 4-hydroxy-L-threonine (via erythroic acid 4-phosphate, 3,4-dihydroxy-2-oxobutanoic acid 4-phosphate, and 4-hydroxy-L-threonine 4-phosphate). This new hypothesis is consistent with the results of the tracer experiments with ¹⁴C-labeled substrates on which the earlier hypothesis had been based (30). The glycolaldehyde/glycine route to 4-hydroxy-L-threonine was regarded as an alternative minor pathway.

We now provide the first direct experimental evidence in support of the ideas of Lam and Winkler.

To prove that 4-hydroxy-L-threonine (13, Scheme 2) lies on the route from glucose into pyridoxol, it had to be demonstrated that its presence in the system in excess spared the incorporation of glucose carbon into the C₃ unit C-6,5,5 of pyridoxol. To prove intact incorporation of 4-hydroxy-L-threonine, it had to be demonstrated that the bond, generated in the formation of the compound from glycolaldehyde and glycine, i.e. the bond destined to become C-5,6 of pyridoxol, remains intact in the course of entry of 4-hydroxy-L-threonine into pyridoxol. Finally, to substantiate the proposal of Lam and Winkler, that the compound originated from glucose via the pentose phosphate route, direct evidence of the intermediacy of a compound related to the proposed route was required.

Evidence that each of these three conditions is fulfilled is provided by Experiments 7, 8, and 9, respectively. The ¹³C NMR spectrum (E, Fig. 3) of the sample of pyridoxol, isolated after administration of [1,2,3,4,5,6-¹³C₆]D-glucose in the presence of excess 4-hydroxy-L-threonine (Experiment 7) shows that the signals due to C-6, ( 131.8), C-5 ( 138.8), and C-5 ( 60.1) appear as singlets, whereas those due to the other five carbon atom all maintain their multiplicity (8). The presence of excess 4-hydroxy-L-threonine had completely suppressed incorporation of glucose-derived ¹³C into the C₃ unit, C-6,5,5 of pyridoxol, while incorporation of glucose-derived ¹³C into the rest of the molecule was unaffected (Scheme 2, E; Fig. 3). Thus, 4-hydroxy-L-threonine lies on the route from glucose into pyridoxol. The ¹³C NMR spectrum (Fig. 5, G) of the sample of pyridoxol, isolated after administration of [2,3-¹³C₂]4-hydroxy-L-threonine (11) (Experiment 8) shows satellites at the signals due to C-5 and C-6 ( 138.8, 131.8, ¹J_5,6 = 64.9 Hz), and nowhere else, proving incorporation of the substrate without cleavage of the C-2,3 bond (9).

G

H

Finally, the ¹³C NMR spectrum (Fig. 5, H) of the sample of pyridoxol, isolated from an experiment with [2,3-¹³C₂]D-erythroic acid (Experiment 9), showed that bond-label had been transferred from the substrate into C-6,5 of pyridoxol. This result represents the first direct evidence in support of the proposal of Lam and Winkler (29). Satellites, which are clearly visible in the expanded spectrum (Fig. 5, H), appear at the signals due to C-5 and C-6 ( 138.8, 131.8, ¹J_5,6 = 64.9 Hz).

The evidence is thus complete that the C₃N unit that serves as the source of N-1,C-6,5,5 of pyridoxol is derived from 4-hydroxy-L-threonine, and that the latter is derived from glucose via erythroic acid. Recent results indicate that the first vitamin B₆ compound to be formed in E. coli is pyridoxol 5-phosphate, rather than pyridoxol (31, 32). Even though the nonphosphorylated compounds, 4-hydroxy-L-threonine and D-erythroic acid, were incorporated intact into the C₃N unit, N-1,C-6,5,5 of pyridoxol, it would appear more likely that 4-hydroxythreonine 4-phosphate (33) and erythroic acid 4-phosphate, rather than the non-phosphorylated compounds, serve as the precursors. The presence in E. coli of a nonspecific kinase would account for the utilization, in our experiments, of the nonphosphorylated compounds.

The status of the alternative source of the C₃N unit N-1,C-6,5,5 of pyridoxol and, presumably, also of its precursor, 4-hydroxy-L-threonine, i.e. its derivation from glycolaldehyde plus glycine, must now be evaluated.

Glycolaldehyde satisfies the nutritional requirement for pyridoxol in the pdxB E. coli B mutant, WG3 (34). The evidence is unequivocal that glycolaldehyde is incorporated into pyridoxal. In this mutant (25), as well as in the pdxH mutant WG2 (26), label from specifically ¹⁴C-labeled glycolaldehyde enters C-5,5. The aldehyde carbon of glycolaldehyde supplies C-5 of pyridoxal and the carbinol carbon supplies C-5 (25, 26). Furthermore, in mutant WG3, the N-C bond of the NH₂-CH₂- group of glycine yields the N-1,C-6 bond of pyridoxal (27). This incorporation pattern is entirely consistent with the notion that 4-hydroxy-L-threonine, the precursor of the C₃N unit N-1,C-6,5,5, is synthesized not only from D-glucose via D-erythroic acid, but also from glycine plus glycolaldehyde (20), by a reaction analogous to that normally catalyzed by serine hydroxymethylase.

Whether or not this alternative derivation of 4-hydroxy-L-threonine is a normal, if minor, source of this compound or whether it is induced only if glycolaldehyde is supplied to the medium is a question that cannot be answered on the basis of available knowledge. However, we incline to the view that, unless further more direct evidence becomes available, the glycolaldehyde/glycine route to 4-hydroxy-L-threonine and thence into the C₃N unit, N-1,C-6,5,5, of pyridoxol should be regarded as an artifact, which is observed only when glycolaldehyde is supplied to the culture medium.

Only five pdx genes, pdxA, pdxB, pdxH, pdxJ, and SerC (pdxF) have been identified in E. coli. The protein product of pdxH is pyridoxol 5-oxidase (EC 1.1.3.13) (31, 32). pdxB codes for the enzyme that oxidises D-erythroic acid 4-phosphate to 2-oxo-D-erythroic acid 4-phosphate (EC 1.1.1.?) (35). The latter compound, in turn, is transaminated to 4-hydroxy-L-threonine 4-phosphate by 3-phosphoserine transaminase (EC 2.6.1.62), the product of SerC (pdxF) (29). The protein products of pdxA and pdxJ are unknown. Since neither pdxA nor pdxJ mutants are supported by 4-hydroxy-L-threonine (28), it can be deduced that pdxA and pdxJ are not implicated in the production of this amino acid. Nor is it likely that these genes are involved in the route to 1-deoxy-D-xylulose, since this substrate is a precursor, in E. coli, not only of pyridoxine but also of thiamin (10), and neither pdxA nor pdxJ mutants require thiamin for growth on a minimal medium. The remaining possibility is that the gene products of pdxA and pdxJ play a role in the reaction sequence that generates pyridoxol from the two intermediates, 4-hydroxy-L-threonine and 1-deoxy-D-xylulose. A biochemically and chemically rational hypothesis for the union of these two substrates to yield pyridoxol has been advanced (30). This reaction sequence and the possible involvement of the gene products of pdxA and pdxJ remain to be substantiated experimentally.