Mechanism of biosynthesis of 2-oxo-3-methylvalerate in Chlorobium vibrioforme

Abstract

The biosynthesis of 2-oxo-3-methylvalerate in Chlorobium vibrioforme was investigated by '3C nuclear magnetic resonance spectroscopy of the oxoacid formed from "3C-labeled acetate by washed suspensions. The threonine pathway could be excluded, and the results are in accord with a mechanism for the formation of 2-oxobutyrate from acetyl coenzyme A and pyruvate via citramalate.The green sulfur bacteria are obligately anaerobic and phototrophic and fix CO2 by a reductive tricarboxylic acid cycle (6,7,9,13 Spectrophotometric determination of total branched-chain 2-oxoacids was carried out as described earlier (18). The individual oxoacids were separated as the trimethylsilyl (TMS) derivatives (11) by gas-liquid chromatography on a column containing 10% OV-17 (Supelco) in Gaschrom Q in a Perkin-Elmer 3920 gas chromatograph equipped with a hydrogen flame ionization detector. The carrier gas was N2, and the column temperature was 100°C. The TMS derivative of malonic acid was used as the internal standard. The 2-oxoisocaproate derivative gave double signals due to isomerization, and the one for C-2 overlapped with the C-2 signal of 2-oxo-3-methylvalerate. The value for the latter therefore had to be determined by calculation from the peak ratios (12).The distribution of 13C in the oxoacids was determined by NMR spectroscopy of the 2,4-dinitrophenylhydrazones, which were isolated in the crystalline state from the supernatant fluid. 13C NMR spectra were obtained on a JEOL FX60 spectrometer operating at 15.0 MHz. The 2,4-dinitrophenylhydrazones (approximately 25 mg) were dissolved in 1.5 ml of tetradeuteromethanol, and TMS was used as the internal standard. The number of pulses accumulated was 36,000, the pulse width was 8 jxs, and the pulse delay was 1.8 s.Chemical shifts of the oxoacid carbons relative to TMS were determined with the derivatives of standard (unlabeled) oxoacids, the assignments being based on off-resonance experiments (Table 1).The 2-oxoisocaproate derivative gave two signals for each C atom. That for C-2 overlapped the C-2 peak of 2-oxo-3-methylvalerate. The value for the latter had therefore to be calculated from the peak ratios as determined on standards.The aromatic signal at 123.8 ppm was used as a reference atom (C1) in the calculation of relative signal strengths. The expression K/Ki refers to the signal strength of carbon atom j using carbon atom i as the reference in the oxoacid derivatives formed from unlabeled CO2 and acetate. The expressions Mj and Mi denote the signal intensities corresponding to carbon atoms j and i, respectively, in the oxoacid derivatives formed from 13C-enriched substrates.The proportions of branched-chain 2-oxoacids formed by C. vibrioforme were determined by gas-liquid chromatography prior to the 13C labeling experiments. The dominant oxoacid was 2-oxoisocaproate, present at about twice the concentration of 2-oxo-3-methylvalerate. Increased light intensity or the presence of acetate slightly stimulated oxoacid formation but had no effect on the ratio of acids formed. A...

Citramalate synthase yields a biosynthetic pathway for isoleucine and straight- and branched-chain ester formation in ripening apple fruit

“…-Acetate Feeding. Previous work in plant and nonplant systems(22)(23)(24)(41)(42)(43)(44)(45) was used to predict differential labeling of citramalate and other metabolites in the citramalate pathway from 1-[ 13 C], 2-[ 13 C], and 1,2-[ 13 C 2 ]-acetate feeding (Fig.1). Consistent…”

Enhanced phenylpyruvic acid production with Proteus vulgaris by optimizing of the fermentation medium

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