A Mutant D‐Fructose‐6‐Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated Compounds

Abstract

A mutant of d-fructose-6-phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The k cat /K M value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5-fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis.

Enzyme‐Catalyzed Aldol Additions

“…Some examples were reported on the synthesis of nitrocyclitols by a tandem aldol addition intramolecular Henry reaction (Scheme 8.30). Interestingly, the aldol addition of DHA to GO proceeded in 80% aldehyde conversion to d-xylulose (Scheme 8.30) [6]. This was the most striking result since using FSA wild type, and d-threose was exclusively formed arising from the homo-aldol addition reaction of GO [106].…”

“…The most important aldolase is the class I N-acetylneuraminic acid aldolase (NeuA, EC 4.1.3.3) (Scheme 8.1), also known as sialic acid aldolase, which has a preference for N-acetyl-d-mannosamine (ManNAc; 2) or configurationally related aldohexose sugars and derivatives as the electrophile. The 2-keto-3-deoxy-d-manno-octosonate aldolase (KdoA, EC 4.1.2.23) (Scheme 8.1) has a preference for d-arabinose (3) as the acceptor, whereas two functionally related enzymes, the 2-keto-3-deoxy-6-phospho-d-gluconate (KDPGlc aldolase or GlcA; EC 4.1.2.14) and 2-keto-3-deoxy-6-phospho-d-galactonate aldolases (KDPGal aldolase or GalA; EC 4.1.2.21), have a preference for d-glyceraldehyde-3-phosphate (6) or other small aldehyde electrophiles. Other less commonly known pyruvate aldolases are described in the following sections.…”

“…In terms of k cat /K M , the preference of FSA A129S for the donor substrate changed to DHA > HA > GO, complementing the synthetic abilities to those of FSA wild type [6]. It is likely that the serine residue allows hydrogen bonding that stabilizes the DHA donor substrate and also might be involved in the stabilization of the Schiff base intermediate [108].…”

“…This facilitates the preference of FSA toward the more hydrophobic substrates HA and HB and diminishes the efficacy for DHA, whereas TalB Phe178Tyr has a strong preference for DHA (Table 8.3). As mentioned earlier, FSA mutant Ala129Ser, designed to resemble the donor binding site of TalB [107], exhibited improved tolerance toward DHA [6]. In the opposite direction, the poor tolerance of TalB Phe178Tyr toward HA could be partially overcome by substituting the hydrophilic Ser176 for the hydrophobic Ala in the active site (i.e., the double mutant TalB Phe178Tyr/Ser176Ala) (Figure 8.7), the hydrophobic equivalent Ala129 in FSA [111].…”

“…However, it is essential to understand the changes produced in order to develop new or improved activities in a rational and efficient way. Site-directed mutagenesis needs information about the role of the amino acid residues involved in the catalysis to propose specific modifications (i.e., structure-guided re-design), and this approach offers a great potential for the rapid preparation of mutants with new/improved activities when the structure of the enzyme structure and its catalytic mechanism are known [1,[3][4][5][6].…”