Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors.

Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.

A high-performance liquid chromatography assay for threonine/serine dehydratase.

Threonine dehydratase, a key enzyme leading to the biosynthesis of isoleucine, catalyzes the production of 2-ketobutyrate from threonine. An uv/fluorometric HPLC assay for threonine dehydratase has been developed that involves derivatization of the 2-ketoacids produced by the enzyme using a specific derivatizing agent, o-phenylenediamine. The derivatized ketoacids can be detected spectrophotometrically or fluorometrically. This novel assay is over 1000-fold more sensitive than the commonly used dinitrophenyl hydrazine derivatization assay. In addition, the HPLC assay allows the identification of the reaction product(s), and can be used to examine the reaction using mixtures of the two substrates, threonine and serine.

The peptide sequences near the bound pyridoxal phosphate are conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.

The blocked amino-terminal residue of rat liver serine dehydratase was shown to be acetylalanine by analysis of an isolated amino-terminal peptide after digestion with acylamino acid-releasing enzyme. Digestion of the borohydride-reduced, carboxymethylated enzyme with lysyl endopeptidase yielded a single epsilon-N-pyridoxyllysine-containing peptide, whose sequence is Met-Asp-Ser-Ser-Gln-Pro-Ser-Gly-Ser-Phe-Lys(Pxy)-Ile-Arg-Gly- His-Leu-Cys(Cm)-Lys. This peptide comprises residues 30-49 of the cDNA-deduced amino acid sequence. The sequence of seven amino acids around the bound pyridoxal phosphate is highly conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.

Novel and sensitive activity stains on polyacrylamide gel of serine and threonine dehydratase and ornithine aminotransferase.

A sensitive activity stain for serine and threonine dehydratase is described. After electrophoresis on thin-layer polyacrylamide gel, a reaction mixture containing L-serine or L-threonine as a substrate, NADH, lactate dehydrogenase and agar was solidified on the gel. The oxidation of NADH was recorded by contact printing on photographic paper with UV light. A novel activity stain for ornithine aminotransferase in the gel is also described. A reaction mixture containing substrates, o-aminobenzaldehyde and agar was solidified on the gel. A yellow color developed in a reaction between pyrroline-5-carboxylate and o-aminobenzaldehyde. The photograph was taken by contact printing. Both of the methods are highly sensitive and can detect less than 0.01 unit (mumol product/min at 37 degrees C) of each enzyme.

[Detection and properties of L-threonine-L-serine dehydratase in human liver]. / Obnaruzhenie i svoistva L-treonin-L-serindegidratazy pecheni cheloveka.

It has been shown that in liver extract of men deceased by different causes, L-threonine and L-serine dehydratase activities probably, belonging to only one enzyme--L-threonine-L-serine dehydratase--are found. Both activities and their ratios depend on K+ concentration both in the buffer used for enzyme extraction and in the reaction medium. Before extraction of active and stable forms of enzyme the liver is to homogenized in a buffer containing 0.15 M KCl. Both enzymatic activities have a pH-optimum at pH 9.6--10.0. It was shown that D-isomers of threonine and serine are not dehydratated and do not inhibit dehydratation of L-isomers. Studies of dependence of L-threonine and L-serine dehydratase reaction rates on temperature showed that at any temperature ranges the energy activation values are higher for the L-threonine dehydratase reaction than for the L-serine dehydratase reaction and that the ratio reaction rates for both reactions depends on temperature.

Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase.

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the 'glycine-cleavage system' was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.

Sheep liver serine-threonine dehydratase. I. Purification and evidence for covalently linked alpha-ketobutyrate as its cofactor.

L-Serine-threonine dehydratase (EC 4.2.1.16) from sheep liver has been obtained as a highly purified preparation as shown by ultracentrifuge studies and analytical disc gel electrophoresis. The dehydratase has a molecular weight of 98,000 +/- 10,000 and is composed of two nonidentical subunits with molecular weights of 41,000 and 47,000. The 41,000 subunit is covalently linked to the carbonyl reagent-sensitive coenzyme which has been identified as alpha-ketobutyric acid.

Purification of biosynthetic threonine deaminase from Escherichia coli.

Biosynthetic threonine deaminase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) was purified to apparent homogeneity from cell extracts of Escherichia coli by chromatographic procedures using valine-Sepharose, isoleucine-N-hexamethyleneamine-Sepharose, and hydroxyapatite with an overall yield of 40%. Analytical ultracentrifugation shows a molecular weight of 214 000. In sodium dodecyl sulfate gel electrophoresis, the enzyme migrates as a single band corresponding to a molecular weight of about 50 000. These data confirm that the enzyme is a tetramer. The sedimentation coefficient, s-020,w, determined by differential sedimentation experiments is 9.2 S. The enzyme shows absorption maxima at 415 and 280 nm. Determination of pyridoxal phosphate by three indenpendent methods shows the presence of two molecules of pyridoxal phosphate per enzyme molecule, the different methods being in excellent agreement equilibrium dialysis experiments establish the presence of two isoleucine binding sites. The Scatchard plot suggests non-cooperativity of these sites. The association constant for isoleucine is 1.2 - 10(5)M-1.