Broadening Deoxysugar Glycodiversity: Natural and Engineered Transaldolases Unlock a Complementary Substrate Space

Abstract

The majority of prokaryotic drugs are produced in glycosylated form, with the deoxygenation level in the sugar moiety having a profound influence on the drug's bioprofile. Chemical deoxygenation is challenging due to the need for tedious protective group manipulations. For a direct biocatalytic de novo generation of deoxysugars by carboligation, with regiocontrol over deoxygenation sites determined by the choice of enzyme and aldol components, we have investigated the substrate scope of the F178Y mutant of transaldolase B, TalB(F178Y), and fructose 6-phosphate aldolase, FSA, from E. coli against a panel of variously deoxygenated aldehydes and ketones as aldol acceptors and donors, respectively. Independent of substrate structure, both enzymes catalyze a stereospecific carboligation resulting in the D-threo configuration. In combination, these enzymes have allowed the preparation of a total of 22 out of 24 deoxygenated ketose-type products, many of which are inaccessible by available enzymes, from a [3×8] substrate matrix. Although aliphatic and hydroxylated aliphatic aldehydes were good substrates, D-lactaldehyde was found to be an inhibitor possibly as a consequence of inactive substrate binding to the catalytic Lys residue. A 1-hydroxy-2-alkanone moiety was identified as a common requirement for the donor substrate, whereas propanone and butanone were inactive. For reactions involving dihydroxypropanone, TalB(F178Y) proved to be the superior catalyst, whereas for reactions involving 1-hydroxybutanone, FSA is the only choice; for conversions using hydroxypropanone, both TalB(F178Y) and FSA are suitable. Structure-guided mutagenesis of Ser176 to Ala in the distant binding pocket of TalB(F178Y), in analogy with the FSA active site, further improved the acceptance of hydroxypropanone. Together, these catalysts are valuable new entries to an expanding toolbox of biocatalytic carboligation and complement each other well in their addressable constitutional space for the stereospecific preparation of deoxysugars.

Computational Tools for Rational Protein Engineering of Aldolases

“…Recently, the substrate scope of FSA and TalB F178Y has been investigated with respect to the synthesis of deoxysugars [93]. This study revealed a complementary donor specificity of the two enzymes.…”

“…By a replacement of A129S in FSA, which resembles the TalB active site, the catalytic efficiency towards DHA was greatly improved (17-fold) [94]. The reciprocal substitution in TalB F178Y (TalB F178Y/S176A) resulted in an increased activity for HA [93]. …”

Enzyme‐Catalyzed Aldol Additions

“…For both enzymes, the hydroxyacetyl portion in the donor is up to now an absolute structural requirement in either or both Schiff base formation and nucleophile generation [111]. The variable moiety of the donor substrates is accommodated in a highly hydrophobic environment in the binding site of FSA (Leu107, Ala129), whereas polar residues are found in the equivalent positions of TalB (Asn154, Ser176) (Figure 8.7).…”

“…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]. The complementary behavior of FSA and TalB mutant was recently exploited to prepare a collection of deoxygenated carbohydrates using FSA for HA and HB aldol additions and TalB Phe178Tyr with DHA (Table 8.4) [6,111].…”

“…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]. The complementary behavior of FSA and TalB mutant was recently exploited to prepare a collection of deoxygenated carbohydrates using FSA for HA and HB aldol additions and TalB Phe178Tyr with DHA (Table 8.4) [6,111]. Interestingly, FSA Ala129Ser and TalB Phe178Tyr render mainly the cross-aldol reaction product of DHA and GO, whereas FSA wild type provides exclusively the self-aldolization product, d-threose [106].…”