L-Rhamnulose 1-phosphate aldolase

Four DHAP converting aldolases are known, these can synthesize different diastereomers with complementary configurations D-fructose (FruA EC 4.1.2.13) and D-tagatose 1,6-bisphos-phate (TagA, F.C 4.1.2.-), L-fuculose (FucA EC 4.1.2.17) and L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19)3. The synthetic application of the first (class 1 or 2) and the latter two types (class 2) has been examined. 

A solution of 2.25 g (25 mmol) of D-glyccraldehyde in 300 mL of water is combined with a solution of 20 mmol of dihydroxyacetonc phosphate (DIIAP) in 200 mL of water freshly adjusted to pH 6.8. The mixture is incubated with 100 U of L-rhamnulose 1-phosphate aldolase at r.t. for 24 h with monitoring of conversion by TLC (2-propanol/sat. ammonia/water 6 4 2) and by enzymatic assay for DHAP55. 

In the case of L-rhamnulose-1-phosphate aldolase (RhaD), we found that the problem of phosphorylated substrate requirement (dihydroxyactone phosphate (DHAP)) could be overcome by a simple change in buffer. Thus, when using borate buffer, reversible borate ester formation created a viable substrate out of dihydroxyacetone, which is not otherwise accepted by the wild-type enzyme (Figure 6.6) [23]. The process was used in a one-step synthesis of... 

The L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19) is found in the microbial degradation of L-rhamnose which, after conversion into the corresponding ketose 1-phosphate 44, is cleaved into 41 and L-lactaldehyde (l-16). The RhuA has been isolated from E. coli [336-339], and characterized as a metallo-protein [194,340,341]. Cloning was reported for the E. coli [342,343] and Salmonella typhimurium [344] genes, and construction of an efficient overexpression system [195,220] has set the stage for crystallization of the homotetrameric E. coli protein for the purposes of an X-ray structure analysis [345]. 

For the described approach, it is important to note that aldolases of different origin were tested and that, in contrast to L-rhamnulose-1-phosphate aldolase (RhuA), the D-fructose-1,6-biphosphate aldolase from rabbit muscle and L-fucu-lose-1-phosphate aldolase from E. coli were not active for DHAP/(R)-N- and (S)-iV-Cbz-prolinal condensation. Since RhuA accepts both, (S)-N- and (R)-N-Cbz prolinals, the chemo-enzymatic synthesis of both, hyacinthacines A and A2 isomers could be achieved. In conclusion, the origin and the particular enzyme itself... 

These systems were tested in the enzymatic aldolization of a variety of A/-Cbz-aminoaldehydes catalyzed by D-fructose-l,6-bisphosphate aldolase from rabbit muscle (RAMA) and L-rhamnulose-1-phosphate aldolase and L-fuculose-1-phosphate aldolase from E. coli (Espelt et al. 2003 a,b, 2005). The largest differences between conventional DMF/water cosolvent systems and gel emulsions were observed with RAMA catalyst (Fig. 6.5.11). 

A combination of glycerol phosphate oxidase (GPO), catalase, and L-rhamnulose-1-phosphate aldolase (RhuA) allowed for an efficient conversion of aminoglycerol into 1-deoxy-l-phosphoramido-L-fructose (Scheme 24). 

Aldol addition of DHAP to aldehydes is catalyzed by DHAP-dependent aldolases. Two stereogenic centers are formed and therefore four possible stereoisomers can be obtained. Although nature has evolved a set of four distinct stereocomplementary types (Scheme 10.3), so far, only three of the known DHAP-dependent aldolases, namely the D-fructose-l,6-bisphosphate aldolase (FruA), L-rhamnulose-1-phosphate aldolase (RhuA), and L-fuculose-1-phosphate aldolase (FucA), have found broad synthetic applicability due to their high stereoselectivity and broad acceptor tolerance [5,77]. DHAP-dependent aldolases are highly selective for the nucleophilic substrate DHAP, tolerating only few isosteric modifications [84-88]. 

Stereocomplemenlary set of DHAP-dependent aldolases. D-Fructose-1,6-bisphosphate aldolase (FruA), L-rhamnulose-1-phosphate aldolase (RhuA), L-fuculose-1-phosphate aldolase (FucA), D-tagatose-1,6-hisphosphate aldolase (TagA)... 

Garrabou, X., Joglar, J., Parella, T., Bujons, J., and Clapes, R, Redesign of the phosphate binding site of L-rhamnulose-1-phosphate aldolase towards a dihydroxyacetone dependent aldolase. Adv. Synth. Catal. 2011,353 (1), 89-99. 

Kroemer, M., Merkel, I., and Schulz, G. E., Structure and catalytic mechanism of L-rhamnulose-1-phosphate aldolase. Biochemistry 2003, 42 (36), 10560-10568. 

---