4-Phospho-d-erythronate is an intermediate in the synthesis of pyridoxal 5′-phosphate in some bacteria and an inhibitor of ribose 5-phosphate isomerase. Previous synthetic schemes for the preparation of 4-phospho-d-erythronate required expensive precursors and typically gave low yields. We report a straightforward synthesis of 4-phospho-d-erythronate from the inexpensive precursor d-erythronolactone in five steps with a preparatively useful yield of 22%.

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-d-erythronate (4PE) to 2-oxo-3-hydroxy-4-phosphobutanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not reoxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-keto acids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ∼1 × 104 M−1 s−1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s−1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M−1 s−1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that d-2-HGA, but not l-2-HGA, is a competitive inhibitor vs 4PE and a noncompetitive inhibitor vs α-ketoglutarate.

Tetrachlorohydroquinone (TCHQ) dehalogenase is profoundly inhibited by its aromatic substrates, TCHQ and trichlorohydroquinone (TriCHQ). Surprisingly, mutations that change Ile12 to either Ser or Ala give an enzyme that shows no substrate inhibition. We have previously shown that TriCHQ is a noncompetitive inhibitor of the thiol−disulfide exchange reaction between glutathione and ESSG, a covalent adduct between Cys13 and glutathione formed during dehalogenation of the substrate. Substrate inhibition of the thiol−disulfide exchange reaction is less severe in the I12S and I12A mutant enzymes, primarily due to weaker binding of TriCHQ to ESSG. These mutations also result in a decrease in the rate of dehalogenation. Because the rate-limiting step in the I12S and I12A enzymes is dehalogenation, rather than the thiol−disulfide exchange reaction, the relatively modest inhibition of the thiol−disulfide exchange reaction does not affect the overall rate of turnover.

Evolution of new enzymatic activities is believed to require a period of gene sharing in which a single enzyme must serve both its original function and a new function that has become advantageous to the organism. Subsequent gene duplication allows one copy to maintain the original function, while the other diverges to optimize the new function. The physiological impact of gene sharing and the constraints imposed by the need to maintain the original activity during the early stages of evolution of a new activity have not been addressed experimentally. We report here an investigation of the evolution of a new activity under circumstances in which both the original and the new activity are critical for growth. Glutamylphosphate reductase (ProA) has a very low promiscuous activity with N-acetylglutamylphosphate, the normal substrate for ArgC (N-acetylglutamylphosphate reductase). A mutation that changes Glu-383 to Ala increases the promiscuous activity by 12-fold but decreases the original activity by 2,800-fold. The impairment in Pro and Arg synthesis results in 14-fold overexpression of E383A ProA, providing sufficient N-acetylglutamylphosphate reductase activity to allow a strain lacking ArgC to grow on glucose. Thus, reaching the threshold level of NAGP reductase activity required for survival required both a structural mutation and overexpression of the enzyme. Notably, overexpression does not require a mutation in the regulatory region of the protein; amino acid limitation attributable to the poor catalytic abilities of E383A ProA causes a physiological response that results in overexpression.

The genetic code has certain regularities that have resisted mechanistic interpretation. These include strong correlations between the first base of codons and the precursor from which the encoded amino acid is synthesized and between the second base of codons and the hydrophobicity of the encoded amino acid. These regularities are even more striking in a projection of the modern code onto a simpler code consisting of doublet codons encoding a set of simple amino acids. These regularities can be explained if, before the emergence of macromolecules, simple amino acids were synthesized in covalent complexes of dinucleotides with α-keto acids originating from the reductive tricarboxylic acid cycle or reductive acetate pathway. The bases and phosphates of the dinucleotide are proposed to have enhanced the rates of synthetic reactions leading to amino acids in a small-molecule reaction network that preceded the RNA translation apparatus but created an association between amino acids and the first two bases of their codons that was retained when translation emerged later in evolution.