Glycine-dependent aldolases

Of the known classes of aldolase, DERA (statin side chain) and pyruvate aldolases (sialic acids) have been shown to be of particular value in API production as they use readily accessible substrates. Glycine-dependent aldolases are another valuable class that allow access to p-hydroxy amino acid derivatives. In contrast, dihydroxy acetone phosphate (DHAP) aldolases, which also access two stereogenic centres simultaneously,... 

In nature, most aldolases are rooted in the sugar metabolic cycle and accept highly functionalized substrates for the aldol reaction. Nevertheless, the scope of enzymatic aldol reactions is limited, since aldolases strictly distinguish between the acceptor and the donor, yielding almost exclusively one product, and is furthermore restricted to only a few different possible natural donors. According to the donor molecules, aldolases are grouped in dihydroxyacetone phosphate-, phosphoenolpyruvate- or pyruvate-, acetaldehyde-, and glycine-dependent aldolases [41]. 

Fig. 35 Synthetic applications of (a) DHAP-, (b) pyruvate-, and (c) glycine-dependent aldolases...

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]. 

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]. 

Scheme 5.2. The four main groups of aldolase reactions classified by their donor substrate (1) Dihydroxyacetone phosphate (DHAP)- dependent aldolases, (2) phosphoenol pyruvate (PEP)-and pyruvate-dependent aldolases, (3) 2-deoxyribose-5-phosphate aldolase (DERA), a member of the acetaldehyde-dependent aldolases, and (4) glycine-dependent aldolases (GDA).

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... 

Scheme 5.47. Glycine-dependent aldolases and the reactions they catalyze.

One important application of glycine-dependent aldolases is for the kinetic resolution of p-hydroxy-a-amino acids [162]. A recent example is the kinetic resolution of p-phenylserine (130), p-(nitrophenyl) serine (131) and p-(methylsulfonylphenyl) serine (132) catalyzed by immobilized E. coli cells overexpressing SHMT (93-97% conversion was obtained with ca 98% ee) (Scheme 10.29). The immobUized cells where reused up to ten times exhibiting an excellent operational stability and an average conversion of 60%. 

Glycine-Dependent Aldolases (ThrA) The group of the glycine-dependent aldolases affords the synthesis of (S-hydroxy-a-amino-acids, d- and L-threonine and serine, most of them are known as L-threonine aldolases (ThrA). They catalyzed the addition of glycine 31 to various aldehyde substrates with pyridoxal-5 -phosphate (PLP) as cofactor. The formed products contain two stereogenic centers whose stereochemistry is controlled by the choice of D- or L-threonine and corresponding d- or L-a//o-threo-nine aldolases (Scheme 28.16). 

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]. 

Serine hydroxymethyltransferase is a PLP-dependent aldolase. It catalyzes interconversion between glycine and various P-hydroxy-a-amino acids, such as serine and threonine, via formation of a quinoid intermediate derived from PLP with the amino acid substrate (Scheme 2.9). This aldolase-type reaction is of interest as an asymmetric synthesis of a-amino acids via C-C bond formation. 

Serine Hydroxymethyltransferase Serinehydroxymethyltrans-ferase is a pyridoxed phosphate-dependent aldolase that catalyzes the cleavage of serine to glycine and methylene-tetrahydrofolate (as shown in Figure 10.5). Serine is the major source of one-carbon substituted folates for biosynthetic reactions. At times of increeised cell proliferation, the activities of serine hydroxymethyltransferase emd the enzymes of the serine biosynthetic pathway cue increased. The other product of the reaction, glycine, is also required in increased cimounts under these conditions (for de novo synthesis of purines). 

A second catabolic reaction of L-threonine (Eq. 24-37, step b) is cleavage to glycine and acetaldehyde. The reaction is catalyzed by serine hydroxymethyl-transferase (Eq. 14-30). Some bacteria have a very active D-threonine aldolase. A quantitatively more important route of catabolism in most organisms is dehydrogenation (Eq. 24-37, step to form 2-amino-3-oxobutyrate. This intermediate can be cleaved by another PLP-dependent enzyme to acetyl-CoA plus glycine (Eq. 24-38, step d). It can also be decarboxylat-ed (Eq. 24-38, step e) to aminoacetone, a urinary excretion product, or oxidized by amine oxidases to methylglyoxal (Eq. 24-37, The latter can... 

In a schematic elution pattern of some standard proteins, peroxidase was eluted first with saline, BSA came next with glycine buffer at pH 6.6 and hemoglobin and catalase were eluted at a pH of nearly 8.0. Aldolase, lysozyme, chymotrypsinogen A, malate dehydrogenase, and cytochrome c were not eluted under these conditions, but were eluted with 0.1% SDS. The adsorption order does not depend on the isoelectric point, the molecular mass, or the content of basic amino acids. However, adsorption may depend on the o -helix content, and the secondary structure of those proteins may be important. We have also reported on protein adsorption and separation on siliconized glass surfaces (30), and on the adsorption and separation of nucleic acids on those same surfaces (31-35). 

In humans, camitine is either obtained from the diet or synthesised de novo (Fig. 1). Camitine biosynthesis in higher eukaryotes starts when protein-bound L-lysine is trimethylated by a protein-dependent methyltransferase to form e-N-trimethyllysine. Upon degradation of these proteins, free e-N-trimethyllysine becomes available and is hydroxylated at the 3-position by e-N-trimethyllysine hydroxylase. Subsequently, P-hydroxy- e-N-trimethyllysine is cleaved into ytrimethylaminobutyraldehyde and glycine by p-hydroxy-e-N-trimethyllysine aldolase, after which the aldehyde is oxidized by y-trimethylaminobutyraldehyde dehydrogenase to yield y-butyrobetaine. Finally, y-buty-robetaine is hydroxylated at the 3-position by "j utyrobetaine hydroxylase (y BBH) to produce L-camitine (see Fig. 1). 

The metabolism of j5-hydroxy-a-amino acids involves pyridoxal phosphate-dependent enzymes, classified as serine hydroxymethyltransferase or threonine aldolases, that catalyze reversible aldol-type cleavage to aldehydes and glycine (134) [284]. 

Classification of aldolases according to their donor selectivity (a) pyruvate aldolases, (b) dihydr-oxyacetone phosphate (DHAP)-dependent aldolases, (c) DHA-and other unphosphorylated analogues or DMA utilizing aldolases, (d) glycine/alanine aldolases, and (e) acetaldehyde-dependent aldolases. 

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... 

Type I and Type II aldolases are also present in the group of pyruvate and acetaldehyde-dependent aldolases (DERA, type I). Only the glycine- and alanine-dependent aldolases work according to a different mechanism involving the PLP cofactor. 

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