Systems Biocatalysis is an emerging concept of organizing enzymes in vitro to construct complex reaction cascades for an efficient, sustainable synthesis of valuable chemical products. The strategy merges the synthetic focus of chemistry with the modular design of biological systems, which is similar to metabolic engineering of cellular production systems but can be realized at a far lower level of complexity from a true reductionist approach. Such operations are free from material erosion by competing metabolic pathways, from kinetic restrictions by physical barriers and regulating circuits, and from toxicity problems with reactive foreign substrates, which are notorious problems in whole-cell systems. A particular advantage of cell-free concepts arises from the inherent opportunity to construct novel biocatalytic reaction systems for the efficient synthesis of non-natural products ("artificial metabolisms") by using enzymes specifically chosen or engineered for non-natural substrate promiscuity. Examples illustrating the technology from our laboratory are discussed.
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.
The four distinct dihydroxyacetone phosphate dependent aldolases". ' I enjoy increasing interest for preparative asymmetric synthesis because of their capacity to build up two new stereogenic centers with high chiral induction.[31 While all DHAP aldolases have a very broad substrate tolerance for the aldol acceptor substrate, they appear to have a high substrate specificity for dihydroxyacetone phosphate (DHAP) as the aldo1 donor, and only few isosteric replacements of the phosphate ester moiety are t~l e r a t e d .~~] Diastereoselectivity may be limited for certain cases in the control of the stereocenter at C-4, which points to occasional inverse binding of the aldehyde carbonyl group.r2ck Certainly, a detailed understanding of the contributions of active site residues in substrate recognition and in the catalytic event is highly desirable to further improve the predictive value of the method.Aldolases have been divided into two classes according t o their mode of donor activation.[51 Class I aldolases achieve stereospecific deprotonation of the substrate by means of covalent linkage to an active site lysine residue (imine/enamine formation), while class I1 aldolases utilize transition metal ions (usually Zn2 +) as essential Lewis acid cofactors to facilitate deprotonation (Fig. 1). For the class I FruA,"] despite extensive efforts using modern techniques of enzymology, site-directed mutagenesis. and protein crystallography,[61 a conclusive model that accounts for the function of the active site residues in the individual steps of catalysis is advancing very slowly.[71 In particular, no structure with bound substrates or inhibitors is yet available.Based on work with the class I1 FruA from yeast, several early hypotheses for the mechanism of Zn-dependent aldolases have been developed. According to ESR and NMR relaxation rate measurements on the Mn'+-substituted holoenzyme, DHAP is bound through its phosphate group,[*] and the carbonyl is polarized by Zn2+ through an intervening imidazole ring (Fig. l , A).'91 Subsequent FT-IR and deuterium exchange studies with native yeast aldolasellO1 led to the conclusion that aldehyde activation occurs by an additional direct coordination of the carbonyl (Fig. 1, B). Recently, Dreyer and Schulz[""I reported the X-ray structure for FucA (2.13 A resolution), the first of a class I1 aldolase. The active site, which is located at the interface between two subunits of the homotetramer, contains the catalytically active Zn2+ ion tightly coordinated by three Ne atoms of histidine residues (His92, His94, His155; Fig. 1, C). Thus, all previous mechanistic hypotheses must be rejected in light of the steric restraints imposed on the Zn2+ ion. which precludes coordination of more than a single substrate, and on its histidine ligands, which cannot act as a proton relay between bound substrates. Under the slightly alkaline conditions required for FucA crystallization, the natural substrate L-fuculose 1-phosphate 1 (Scheme 1) cannot be used for X-ray analysis of the enzyme-ligand comple...
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