Deoxy-D-ribose 5-phosphate aldolase

Natural aldol reaction catalyzed by RibA, and acceptance of non-natural aldol donors. 

Substrate tolerance of deoxy-D-ribose-5-phosphate aldolase. 

Two-stage aldolase-based technical process for deoxyribonucleoside synthesis. 

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

One limitation of this methodology is that DERA is inactivated by high aldehyde concentrations of chloroacetaldehyde. Therefore, industrial applications needed the development of an engineered DERA variant, robust enough to ensure an efficient transformation process [198]. The DSM team applied directed evolution and 

RibA catalyzed the reversible aldol addition of acetaldehyde to D-glyceraldehyde-3-phosphate. 

10 RECENT ADVANCES IN ENZYME-CATALYZED ALDOL ADDITION REACTIONS 

One-pot double addition of acetaldehyde to an aldehyde catalyzed by DERA and two consecutive aldol additions of acetaldehyde and dihydroxyac-etone phosphate (DHAP) catalyzed by a tandem of DERA and FruA catalysts. 

Double addition of acetaldehyde to chloroacetaldehyde and follow-up chemistry for the synthesis of the statin chiral building blocks. Follow-up chemistry (a) Br, H O, pH 5-6, 

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Mechanistically similar to the pyruvate lyases, 2-deoxy-D-ribose 5-phosphate aldolase (EC 4.1.2.4) catalyzes the addition of acetaldehyde to D-glyceraldehyde 3-phosphate. 

Jennewein, S., Schurmann, M., Wolberg, M. et al. (2006) Directed evolution of an industrial biocatalyst 2-deoxy-D-ribose 5-phosphate aldolase. Biotechnology Journal, 1, 537-548. 

Figure 6.2 Overview of biocatalytic routes to vastatin side chains. PLE pig-liver esterase, ADH alcohol dehydrogenase, HHDH halohydrin dehalogenase, DERA 2-deoxy-D-ribose 5-phosphate aldolase.

Another promising route was reported in patent and open hterature by both DSM and Diversa [13, 14]. This route employs a 2-deoxy-D-ribose 5-phosphate aldolase (DERA) that catalyzes a tandem aldol addition in which two equivalents of acetaldehyde (AA) are added in sequence to chloroacetaldehyde (CIAA) to produce a lactol derivative that is similar to the 3,5-dihydoxy side chain of synthetic statins (Figure 6.2e). Diversa screened environmental libraries for novel wild-type DERAs and identified an enzyme that was both tolerant to increased substrate concentrations and more active than DERA from E. coli in the target reaction [13]. 

Deoxy-D-Ribose 5-Phosphate Aldolase (DERAj-Based Routes to Statin Intermediates 1131... 

To date, 2-deoxy-D-ribose 5-phosphate aldolase (DERA) is the only acetaldehyde-dependent aldolase being applied in organic synthesis. Thus the stereoselectivity of DERA is significant, all known enzymes from different organisms showing the same preferences, limiting the field of application to syntheses in which specifically the DERA-catalyzed enantiomer is needed. 

There is only a single literature example of a bi-directional skeleton elongation performed with an enzyme different from the DHAP dependent aldolases. The 2-deoxy-D-ribose 5-phosphate aldolase (RibA EC 4.1.2.4), a bacterial class I enzyme which requires acetaldehyde as the nucleophilic substrate [42], has been applied to the tandem aldolization of a thioether dialdehyde [95]. The substrate 14 was synthesized from optically homogenous (R)-glycidaldehyde and, because of its Q Synimetry, diastereoselective twofold addition of acetaldehyde led to a symmetrical 5,5 -sulfide-linked dipentofuranose 15. 

An economically viable alternative to the synthesis of deoxyribonuclosides has been developed as a two stage process involving 2-deoxy-D-ribose 5-phosphate aldolase (DERA) (Fig. 6.5.14) (Tischer et al. 2001). The first step was the aldol addition of G3P to acetaldehyde catalyzed by DERA. G3P was generated in situ by a reverse action of EruA on L-fructose-1,6-diphosphate and triose phosphate isomerase which transformed the DHAP released into G3P. In a second stage, the action of pentose-phosphate mutase (PPM) and purine nucleoside phosphorylase (PNP), in the presence of adenine furnished the desired product. The released phosphate was consumed by sucrose phosphorylase (SP) that converts sucrose to fructose-1-phosphate, shifting the unfavorable equilibrium position of the later reaction. 

The 2-deoxy-D-ribose 5-phosphate aldolase (RibA or DERA EC 4.1.2.4) is a class I enzyme that, in vivo, catalyzes the reversible addition of acetaldehyde to D-glyceraldehyde 3-phosphate (34 Figure 5.57) in the metabolic degradation of 127 from deoxyribonucleosides [269], ivith an equilibrium constant for synthesis of 2 x lO m [56]. It is, therefore, unique among the aldolases in that it uses an aldehyde rather than a ketone as the aldol donor. RibA has been isolated from eukaryotic and prokaryotic sources [270, 271],... 

Self- and Cross-Aldol Reactions Catalyzed by 2-Deoxy-D-ribose 5-phosphate aldolase... 

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