Threonine aldolases

Wild-type FSA and FSA A129S variant catalyzed synthesis of D-threose, o-xylulose, and 1-deoxy-D-xylulose. 

One-pot synthesis of o-glycero-D-altro-2-octulose 8-phosphate (98). Abbreviations RPE ribu-lose-5-phosphate epimerase RPI ribose-5-phosphate isomerase. 

The pyridoxal-5 -phosphate-dependent threonine aldolases (ThrA EC 4.1.2.5) and serine hydroxymethyltransferase (SHMT EC 2.1.2.1) catalyze the aldol addition of glycine to aldehydes with the formation of two new stereogenic centers [15,44,161-163]. Hence, four possible products can be formally obtained from a single aldehyde, depending on the specificity of the threonine aldolase. All glycine aldolases assayed 

10 RECENT ADVANCES IN ENZYME-CATALYZED ALDOL ADDITION REACTIONS 

Set of accessible threonine aldolases for adol addition of glycine to aldehydes. Abbreviations PLP=Pyridoxal-5 -phosphate. 

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Ah + H2N OH (2R JS)-2-amino-3-hydroxypentanoir acid D-threonine aldolase "f COOH OH 6... 

Figure 10.45 Aldol reactions catalyzed in vivo by serine hydroxymethyl transferase and by threonine aldolases.

The metabolism of P-hydroxy-a-amino adds involves pyridoxal phosphate-dependent enzymes, dassified as serine hydroxymethyltransferase (SHMT) (EC 2.1.2.1) or threonine aldolases (ThrA L-threonine selective = EC 4.1.2.5, L-aHo-threonine selective = EC 4.1.2.6). Both enzymes catalyze reversible aldol-type deavage reactions yielding glycine (120) and an aldehyde (Eigure 10.45) [192]. 

Whereas SHMT in vivo has a biosynthetic function, threonine aldolase catalyzes the degradation of threonine both l- and D-spedfic ThrA enzymes are known [16,192]. Typically, ThrA enzymes show complete enantiopreference for their natural a-D- or a-t-amino configuration but, with few exceptions, have only low specificity for the relative threo/erythro-configuration (e.g. (122)/(123)) [193]. Likewise, SHMT is highly selective for the L-configuration, but has poor threo/erythro selectivity [194]. For biocatalytic applications, the knovm SHMT, d- and t-ThrA show broad substrate tolerance for various acceptor aldehydes, notably induding aromatic aldehydes [193-196] however, a,P-unsaturated aldehydes are not accepted [197]. For preparative reactions, excess of (120) must compensate for the unfavorable equilibrium constant [34] to achieve economical yields. 

Threonine catabohsm merges with that of glycine after threonine aldolase cleaves threonine to glycine and acetaldehyde. 

A classical approach to driving the unfavorable equilibrium of an enzymatic process is to couple it to another, irreversible enzymatic process. Griengl and coworkers have applied this concept to asymmetric synthesis of 1,2-amino alcohols with a threonine aldolase [24] (Figure 6.7). While the equilibrium in threonine aldolase reactions typically does not favor the synthetic direction, and the bond formation leads to nearly equal amounts of two diastereomers, coupling the aldolase reaction with a selective tyrosine decarboxylase leads to irreversible formation of aryl amino alcohols in reasonable enantiomeric excess via a dynamic kinetic asymmetric transformation. A one-pot, two-enzyme asymmetric synthesis of amino alcohols, including noradrenaline and octopamine, from readily available starting materials was developed [25]. 

Other aldolases, from microorganisms, have been cloned and overexpressed. For instance, L-threonine aldolase from Escherichia coli and D-threonine aldolase from Xanthomonus orysae have been obtained and used to prepare 0-hydroxy-a-amino acid derivatives1122. ... 

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). 

Researchers at the University of Graz, in collaboration with scientists from DSM, have developed an elegant and novel approach to the synthesis of P-amino alcohols using two different enzymes in one pot (Scheme 2.35). For example, a threonine aldolase-catalyzed reaction was initially used, under reversible conditions, to prepare L-70 from glycine 69 and benzaldehyde 68. L-70 was then converted to (R)-71 by an irreversible decarboxylation catalyzed by L-tyrosine decarboxylase. In a second example, D/L-syn-70 was converted to (R)-71 using the two enzymes shown combined with a D-threonine aldolase in greater than 99% e. e. and 67% yield ]37, 38]. 

Scheme 2.35 Combined use of threonine aldolase and L-tyrosine decarboxylase.

Marshall, V. M. and Cole, W. M. 1983. Threonine aldolase and alcohol dehydrogenase activities in Lactobacillus bulgaricus and Lactobacillus acidophilus and their contribution to flavour production in fermented foods. J. Dairy Res. 50, 375-379. 

Serine hydro xymethyltransferase Threonine aldolase 8-Aminolevulinate synthase Serine palmitoyltransferase 2-Amino-3-oxobutyrate-CoA ligase... 

An intermediate analogous to that in figure 10.4/7 but generated from glycine and so lacking the /3 and y carbons, can react as a carbanion with an aldehyde to produce a /3-hydroxy-a-amino acid. These reactions are catalyzed by aldolases, such as threonine aldolase or serine hydroxymethyl transferase. 

In various mammalian tissues an enzymatic activity has been reported [451-453] which causes the liberation of glycine 149 and acetaldehyde from L-threonine 150 and has therefore been named threonine aldolase (ThrA EC 4.1.2.5). It is curious that a//o-threonine 151 seems to be a more active substrate for this enzyme than is 150. Cleavage of L-3-phenylserine is also catalyzed by mammalian ThrA enzymes [454-456], which in direction of synthesis nonselec-tively produce both threo (152) and erythro (153) configurated adducts from benzaldehyde and 149 [454], The same enzyme preparations were also able to act on 150. Thus, considerable disagreement still exists in the literature about the true nature of these enzymatic activities. 

Glycine-dependent threonine aldolases have been used to synthesize a number of /-halogenated and long-chain fi-hydroxy-a-amino acids. For D-threonine aldolase. vvn-selectivity was observed exclusively. Further chemical conversion yielded the 2-amino-l,3-diols, potential precursors for the synthesis of short-chain sphingo-sine-derivatives (Fig. 35c) [193]. 

Steinreiber J, Fesko K et al (2007) Synthesis of y-halogenated and long-chain P-hydroxy-a-amino acids and 2-amino-l,3-diols using threonine aldolases. Tetrahedron 63 8088-8093... 

The glycine-dependent aldolases contain a cofactor pyridoxal phosphate (PLP). Binding of glycine to it as an imine enables the deprotonation necessary for the carbon-carbon bond forming reaction, with pyridine acting as an electron sink. The subsequent 100% atom efficient reaction with an aldehyde establishes the new bond and two new stereocenters (Scheme 5.30). Of all the glycine-dependent aldolases only L-threonine aldolase (LTA) is commonly used [40, 43, 52]. 

The glycine-dependent aldolases are pyridoxal 5-phosphate dependent enzymes that catalyze the reversible aldol reaction, where glycine and an acceptor aldehyde form a (i-hydroxy-a-amino acid (Scheme 5.47).74 Serine hydroxymethyltransferases, SHMT (EC 2.1.2.1), and threonine aldolases, two types of glycine dependent aldolases, have been isolated. In... 

L-Threonine Aldolase (LTA)-Catalyzed Synthesis of L- 3-Hydroxy-a-Amino Acids... 

Scheme 5.49. Resolution of fi-hydroxy-a-amino acids, using threonine aldolase from Strepto-myces amakusaensis.

Scheme 5.50. Synthesis of sialyl Lewis X mimetics using L-threonine aldolase (LTA).

Scheme 5.51. L-Threonine aldolase approach to mycestericin D. X, Rj, R2 = protecting groups.

Scheme 5.52. Structure of vero-toxin recognized RNA sequence (left) and preparation of its peptidic mimetic (right) using LTA (L-threonine aldolase).

Loss of side chain aC— 3C Threonine aldolase Serine hydroxymethyltransferase... 

Figure 23.12. Bond Cleavage by PLP Enzymes. Pyridoxal phosphate enzymes lahilize one of three bonds at the a-carhon atom of an amino acid substrate. For example, bond a is labilized by aminotransferases, bond b by decarboxylases, and bond c by aldolases (such as threonine aldolases). PLP enzymes also catalyze reactions at the ()- and y-carbon atoms of amino acids. 

Enzyme-catalyzed reactions in this area include reaction with glycine catalyzed by L-threonine aldolase to afford 164 <2000SL1046> and the use of almond oxynitrilase to catalyze the formation of cyanohydrin 165 by reaction of 161 with acetone cyanohydrin <2001T2213>. 

W. Stbcklein and H.-C. Schmidt, Evidence for L-threonine cleavage and allo-threonine formation by different enzymes from Ciostridium pasteurianium Threonine aldolase and serine hydroxymethyltransferase, Biodiem. ]., 232 621 (1985). 

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