Enzyme-catalyzed aldol addition aldehyde substrates

Literally hundreds of aldehydes have so far been tested successfully by enzymatic assay and preparative experiments as a replacement for 34 in rabbit muscle FruA catalyzed aldol additions [18, 25], and most of the corresponding aldol products have been isolated and characterized. A compilation of selected typical substrates and their reaction products is provided in Table 5.4, and further examples are indicated in Section 5.4.5. In comparison, metal dependant FruA enzymes are more specific for phosphorylated substrates and accept non-phosphorylated substrate analogs only vith much reduced activity (< 1%). 

Aldolases are part of a large group of enzymes called lyases and are present in all organisms. They usually catalyze the reversible stereo-specific aldol addition of a donor ketone to an acceptor aldehyde. Mechanistically, two classes of aldolases can be recognized [4] (i) type I aldolases form a Schiff-base intermediate between the donor substrate and a highly conserved lysine residue in the active site of the enzyme, and (ii) type II aldolases are dependent of a metal cation as cofactor, mainly Zn, which acts as a Lewis acid in the activation of the donor substrate (Scheme 4.1). 

Functionally and mechanistically reminiscent of the pyruvate lyases, the 2-deoxy-D-ribose 5-phosphate (121) aldolase (RibA EC 4.1.2.4) [363] is involved in the deoxynucleotide metabolism where it catalyzes the addition of acetaldehyde (122) to D-glyceraldehyde 3-phosphate (12) via the transient formation of a lysine Schiff base intermediate (class I). Hence, it is a unique aldolase in that it uses two aldehydic substrates both as the aldol donor and acceptor components. RibA enzymes from several microbial and animal sources have been purified [363-365], and those from Lactobacillus plantarum and E. coli could be induced to crystallization [365-367]. In addition, the E. coli RibA has been cloned [368] and overexpressed. It has a usefully high specific activity [369] of 58 Umg-1 and high affinity for acetaldehyde as the natural aldol donor component (Km = 1.7 mM) [370]. The equilibrium constant for the formation of 121 of 2 x 10M does not strongly favor synthesis. Interestingly, the enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates propional-dehyde 111, acetone 123, or fluoroacetone 124 can replace 122 as the donor [370,371], and a number of aldehydes up to a chain length of 4 non-hydrogen atoms are tolerated as the acceptor moiety (Table 6). 

Concerning the reaction S5rstems, hydrophobic aldehydes need cosolvents to enhance their solubility and up to 20% (v/v) dimethylformamide (DMF) or dimethylsulfoxide (DMSO) is usually tolerated by the enzyme without compromising its activity. Emulsions are alternative systems for hydrophobic acceptors, but substrate partitioning between phases may cause rate limitations, particularly for substrates with high values [96]. Examples of aldol additions catalyzed... 

Several DHAP aldolases having different stereospecificities were tested for their acceptance of this phosphonomethyl substrate mimic as the aldol donor, individual enzymes belonging to both Glass 1 and 11 types were found to catalyze the stereoselective addition of 14 to various aldehydes, providing bio-isosteric non-hydrolyzable analogues of sugar 1-phosphates in high yields (for example, 16/17 Scheme 2.2.5.7) [25, 26]. 

Deoxyribose-5-Phosphate Aldolase. The only aldolase known so far which accepts acetaldehyde as donor is 2-deoxyribose-5-phosphate (DER) aldolase. In vivo, DER aldolase catalyzes the reversible aldol reacticm of acetaldehyde and D-glyceraldehyde-3-phosphate to form 2-deoxyribose-5-phosphate. This aldolase is unique in that it is the only aldolase that condenses two aldehydes (instead of a ketone and an aldehyde) to form aldoses (Schemes 2.183 and 2.194). Interestingly, the enzyme (which has been overproduced [1438]) shows a relaxed substrate specificity not only on the acceptor side, but also on the donor side. Thus, besides acetaldehyde it accepts also acetone, fluoroacetone and propionaldehyde as donors, albeit at a much slower rate. Like other aldolases, it transforms a variety of aldehydic acceptors in addition to D-glyceraldehyde-3-phosphate. 

A -acetyl-D-neuraminic acid is biosynthesized from A -acetyl-D-mannosamine and phosphoenol pyruvate, catalyzed by N-acetyl-D-neuraminic acid synthase. The first step involves the addition of an electron pair from the double bond of the phosphoenol pyruvate to the aldehyde group to give an aldol-type condensation (see Fig. 10.8A). The product is the nine-carbon sugar acid, A -acetyl-D-neuraminic acid [23]. In some instances the enzyme requires A-acetyl-D-mannosamine-6-phosphate as the substrate and forms A-acetyl-D-neuraminic acid-9-phosphate. Various hydroxyl groups on C-4, -7, -8, and -9 can be acetylated by specific acetyl transferases using acetyl CoAas the donor. KDO (2-keto-3-deoxy-D-mannooctulosonic acid) is biosynthesized by a very similar condensation between D-arabinose-5-phosphate and pyruvic acid, catalyzed by KDO synthase (see Fig. 10.8B) [24]. 

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