Threonine dehydratase and

A similar stereochemical question as in the /8-replacement reactions can be asked in the a, /8-eliminations where the group X is replaced by a hydrogen, i.e., is the proton added at C-/8 of the PLP-aminoacrylate on the same face from which X departed or on the opposite face This question has been answered for a number of enzymes which generate either a-ketobutyrate or pyruvate as the keto acid product. Crout and coworkers [119,120] determined the steric course of proton addition in the a,/8-elimination of L-threonine by biosynthetic L-threonine dehydratase and of D-threonine by an inducible D-threonine dehydratase, both in Serratia marcescens. Either substrate, deuterated at C-3, was converted in vivo into isoleucine, which was compared by proton NMR to a sample prepared from (3S)-2-amino[3-2H]butyric acid. With both enzymes the hydroxyl group at C-3 was replaced by a proton in a retention mode. Although this has not been established with certainty, it is likely that both enzymes, like other bacterial threonine dehydratases [121], contain PLP as cofactor. Sheep liver L-threonine dehydratase, on the other hand, is not a PLP enzyme but contains an a-ketobutyrate moiety at the active site [122], It replaces the hydroxyl group of L-threonine with H in a retention mode, but that of L-allothreonine in an inversion mode [123]. Snell and coworkers [124] established that the replacement of OH by H in the a, /8-elimination of D-threonine catalyzed by the PLP-containing D-serine dehydratase from E. coli also proceeds in a retention mode. They... 

Although the tryptophan synthetase and tryptophanase reactions have been the best studied replacement and 0 eUmination-deamination reactions, others pf special interest are D-serine dehydratase [75-77] from E. coli, D-threonine dehydratase and L-threonine dehydratase from Serratia marcescens [78]. The only information available on the above enzymes is that in these cases also, the events at occur with retention of configuration. 

One of the most distinguishing features of metabolic networks is that the flux through a biochemical reaction is controlled and regulated by a number of effectors other than its substrates and products. For example, as already discovered in the mid-1950s, the first enzyme in the pathway of isoleucine biosynthesis (threonine dehydratase) in E. coli is strongly inhibited by its end product, despite isoleucine having little structural resemblance to the substrate or product of the reaction [140,166,167]. Since then, a vast number of related... 

Guillouet S, Rodal AA, An G-H, Lessard PA, Sinskey AJ. (1999) Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production. Appl Environ Microbiol 65 3100-3107. 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

L-Serine dehydratase [EC 4.2.1.13], also known as serine deaminase and L-hydroxyaminoacid dehydratase, catalyzes the pyridoxal-phosphate-dependent hydrolysis of L-serine to produce pyruvate, ammonia, and water. In a number of organisms, this reaction is also catalyzed by threonine dehydratase. 

One of the first known examples of allosteric feedback inhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoleucine in five steps (Fig. 6-28). In this system, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition. Isoleucine is quite specific as an inhibitor. No other intermediate in this sequence inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited by isoleucine. Isoleucine binds not to the active site but to another specific site on the enzyme molecule, the regulatory site. This binding is noncovalent and readily reversible if the isoleucine concentration decreases, the rate of threonine dehydration increases. Thus threonine dehydratase activity responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine. 

FIGURE 6-28 Feedback inhibition. The conversion of L-threonine to L-isoleucine is catalyzed by a sequence of five enzymes (E, to E5). Threonine dehydratase (E,) is specifically inhibited allosterically by L-isoleucine, the end product of the sequence, but not by any of the four intermediates (A to D). Feedback inhibition is indicated by the dashed feedback line and the symbol at the threonine dehydratase reaction arrow, a device used throughout this book. 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

There are two types of control shown by allosteric enzymes. They can be modulated by a molecule other than a substrate of the enzyme (termed heterotrophic enzymes, e.g. threonine dehydratase), or by the substrate itself (termed homotrophic enzymes, e.g. oxygen binding to haemoglobin). They contain two or more binding sites for the substrate, and activity is modulated by the number of binding sites which are filled. 

Pyridoxal Phosphate Reaction Mechanisms Threonine can be broken down by the enzyme threonine dehydratase, which catalyzes the conversion of threonine to a-ketobutyrate and ammonia. The enzyme uses PLP as a cofactor. Suggest a mechanism for this reaction, based on the mechanisms in Figure 18-6. Note that this reaction includes an elimination at the j8 carbon of threonine. 

This reaction is readily reversible. Another means of metabolizing serine, which accounts for its glucogenic character, as well as that of glycine, is the conversion of serine to pyruvate, as indicated in Figure 20.12. This reaction is catalyzed by serine dehydratase. A similar enzyme, threonine dehydratase, converts threonine to a-ketobutyrate, and the latter is then converted to propionyl-CoA, as indicated in Figure 20.13. Another similar enzyme, cysteine desulfhydrase, con-... 

Trapping of the aminoacrylate intermediate in the reactions catalyzed by cystathionine-y-synthase and y-cystathionase produced the same diastereomer of KEDB which was different from the one formed with bacterial L-threonine dehydratase. Unfortunately, this experiment has apparently not been done with threonine synthetase. 

Threonine dehydratase catalyzes the formation of 2-ketobutyrate from threonine, and pyruvate from serine. This assay can be used to examine the reaction in the presence of both substrates. 

Serine Racemase (EC 5.1.1.16] Serine racemases have been discovered in both bacteria and eukaryotes (for a review see [60, 62). In the latter organisms, serine racemase catalyzing the conversion of L-Ser to D-Ser was at first discovered in the silkworm Bombyx mori it is a PLP-dependent racemase which is also active on L-Ala (-6% of the activity on L-Ser). A serine racemase was also purified from rat brain (and a serine racemase cDNA was cloned from mouse brain). Mammalian serine racemase shows sequence simUarily with L-threonine dehydratase from various sources all the active site residues of the latter enzyme are also conserved in mouse serine racemase. Mammalian serine racemase is a member of the fold-type II group of PLP enzymes (similarly to L-threonine dehydratase, D-serine dehydratase, and so on) and distinct from alanine racemase, which belongs to the fold-type III group. Mouse serine racemase shows a low kinetic efficiency the Km values for L- and D-Ser are -10 and 60 mM, respectively and the V ax values with L- and D-Ser are 0.08 and 0.37 units/mg protein (less than 0.1% of those of alanine racemase on L- and D-Ala, see above). 

Although the nitrogen atoms of most amino acids are transferred to a-ketoglutarate before removal, the a-amino groups of serine and threonine can be directly converted into NH4 +. These direct deaminations are catalyzed by serine dehydratase and threonine dehydratase, in which PLP is the prosthetic group. 

A classic example of allosteric inhibition is the case of the enzymatic conversion of L-threonine into L-isoleucine by bacteria. The first of five en mes, threonine dehydratase is inhibited by the end product, isoleucine. This inhibition is very specific, and is accomplished only by isoleucine, which binds to a site on the enzyme molecule called the regulatory, or allosteric, site. This site is different from the active site of the en2yme, which is the site of the catalytic action of the eri2yme on the substrate, or molecule being acted on by the eri2yme. 

L-Serine and L-threonine dehydratases dehydrate and subsequently deaminate the amino add to the corresponding a-keto add. These enzymes are known to require pjn-idoxal-S -phosphate as a coenzyme. They can function in a biosynthetic or catabolic marmer (99). Both enzymes can cause problems for the whole-cell-based production of L-serine (100). 

Threonine can be broken down by two separate pathways. Serine dehydratase catalyzes the conversion of threonine to 2-ketobut ate plus an ammonium ion 2-ketobutyrate is then converted by branched-chain keto add (BCKA) dehydrogenase to propionyl-CoA plus carbon dioxide. Propionyl-CoA catabolism is described later in this chapter. Threonine can also be broken down by a complex that has been suggested to be composed of threonine dehydrogenase and acetoacetone synthase (Tressel et ah, 1986). Here, threonine catabolism results in the production of acetyl-CoA plus glycine. 

Serine and threonine dehydrases. Serine and threonine are not substrates in transamination reactions. Their amino groups are removed by the pyridoxal phosphate-requiring hepatic enzymes serine dehydratase and threonine dehydratase. The carbon skeleton products of these reactions are pyruvate and a-keto-butyrate, respectively. 

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