Pyridoxal phosphate enzymes evolution

H. C. Dunathan and J. G. Voet (1974), Stereochemical evidence for the evolution of pyridoxal-phosphate enzymes of various function from a common ancestor. Proc. Nat. Acad. Sci. USA 71, 3888-3891. 

Aspartate aminotransferase is the prototype of a large family of PLP-dependent enzymes. Comparisons of amino acid sequences as well as several three-dimensional structures reveal that almost all transaminases having roles in amino acid biosynthesis are related to aspartate aminotransferase by divergent evolution. An examination of the aligned amino acid sequences reveals that two residues are completely conserved. These residues are the lysine residue that forms the Schiff base with the pyridoxal phosphate cofactor (lysine 258 in aspartate aminotransferase) and an arginine residue that interacts with the a-carboxylate group of the ketoacid (see Figure 23.11). 

This enzyme is a pyridoxal phosphate protein which can also catalyse reactions with hydroxyphenylalanine and 3-hydroxyphenylserine. It catalyses the formation of dopamine which is the precursor of noradrenaline. It may be assayed radiochemically utilising the evolution of C labelled CO2. 

This enzyme is a pyridoxal phosphate protein, which catalyses the formation of 7-aminobutyric acid, a possible chemical transmitter in the CNS. It can be used in an indicator reaction for the radiochemical assay of transaminases. It is assayed radiochemically using Clabelled glutamate and COj evolution [391]. 

The best method for the assay of pyridoxal phosphate is the use of tyrosine decarboxylase as described by Gunsalus, Bellamy, and Umbreit. The enzyme is prepared from a dried powder of cells of S. faecalis R. which has been grown deficient in vitamin Be by growth In a vitamin-Be-free alanine-rich medium. Thus, the decarboxylase is obtained almost completely resolved. This is a convenient preparation, since such a powder is stable for long periods and since the resolution of transaminases, decarboxylases, and tryptophanases isolated from tissues is a rather difficult task. The assay is performed manometrically by measuring the rate of CO2 liberation from tyrosine by the dried powder in the presence of pyridoxal phosphate. The rate of CO2 evolution is a function of the concentration of pyridoxal phosphate. 

Ornithine Decarboxylase Assays. The double-chamber assay system of Moskal and Basu (59) was used to measure enzyme activity in the form of L C] carbon dioxIHe evolution. The assay conditions of O Brien and Diamond (60) were used and consisted of the following components (in micromoles, unless otherwise stated) in a total volume of 100 jul sodium phosphate buffer, pH 7.2, 5.0 EDTA, 1.0 dithiothreitol, 5.0 pyridoxal-5 -monophosphate, 0.2 L-ornithine (specific activity 0.5 x 10° cpm/-jumole), 0.1 and protein, 0.1-0.5 mg. Incubations were carried out at 37°C for 60 min, and the reactions were terminated by the addition of 200 jul of 2M sodium citrate followed by a post-incubation period of 3 hours at 37°C to insure maximal release of radiolabeled carbon dioxide. 

Mehta PK and Christen P (2000) The molecular evolution of pyridoxal-5 -phosphate-dependent enzymes. Advances in Enzymology and Related Areas of Molecular Biology... 

Several coenzymes are involved in the biosynthesis of their own precursors. Thus, thiamine is the cofactor of the enzyme that converts 1-deoxy-D-xylulose 5-phosphate (43) (the precursor of thiamine pyrophosphate, pyridoxal 5 -phosphate and of iso-prenoids via the nomnevalonate pathway) into 2 C-methyl-D-erythritol 4-phosphate (90, Fig. 11). Similarly, two enzymes required for the biosynthesis of GTP, which is the precursor of tetrahydrofolate, require tetrahydrofolate derivatives as cofactors (Fig. 3). When a given coenzyme is involved in its own biosynthesis, we are faced with a hen and egg problem, namely how the biosynthesis could have evolved in the absence of the cmcially required final product. The answers to that question must remain speculative. The final product may have been formed via an alternative biosynthetic pathway that has been abandoned in later phases of evolution or that may persist in certain organisms but remains to be discovered. Alternatively, the coenzyme under study may have been accessible by a prebiotic sequence of spontaneous reactions. An interesting example in this respect is the biosynthesis of flavin coenzymes, in which several reaction steps can proceed without enzyme catalysis despite their mechanistic complexity. 

The general arguments about the antiquity of cofactors apply to PLP. The nonenzymatic synthesis of pyridoxal under prebiotic conditions is considered possible, whereas the presence of a 5 phosphate group could hint to an ancestral attachment of the cofactor to RNA molecules. " Furthermore, there are specific grounds to assume that PLP arrived on the evolutionary scene before the emergence of proteins. In fact, in current metabolism, PLP-dependent enzymes play a central role in the synthesis and interconversion of amino acids, and thus they are closely related to protein biosynthesis. In an early phase of biotic evolution, free PLP could have played many of the roles now fulfilled by PLP-dependent enzymes, since the cofactor by itself can catalyze (albeit at a low rate) reactions such as amino acid transaminations, racemizations, decarboxylations, and eliminations. " This suggests that the appearance of PLP may have preceded (and somehow eased) the transition from primitive RNA-based life forms to more modern organisms dependent on proteins. 

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