Biocatalytic synthesis of hydroxylated natural products using aldolases and related enzymes.

Synthetic building blocks bearing hydroxylated chiral centers are important targets for biocatalysis. Many C-C bond forming enzymes have recently been investigated for new applications and new strategies towards the synthesis of natural products and related oxygenated compounds. Several old catalysts have been studied to increase our functional knowledge of natural aldolase-type enzymes, and new mutated catalysts or catalytic antibodies have been tested for their synthetic utility.

The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution.

Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase.

Catalytic action of fuculose 1-phosphate aldolase (class II) as derived from structure-directed mutagenesis.

Previous analyses established the structures of unligated L-fuculose 1-phosphate aldolase and of the enzyme ligated with an inhibitor mimicking the substrate dihydroxyacetone phosphate. These data allowed us to suggest a catalytic mechanism. On the basis of this proposal, numerous mutations were now introduced at the active center and tested with respect to their catalytic rates and their product distributions. For several mutants, the structures were determined. The results demonstrate the catalytic importance of some particular residues in defined conformations and in the mobile C-terminal chain end. Moreover, they led to a modification of the proposed mechanism. The effect of some mutations on enantioselectivity and on the ratio of diastereomer formation indicates clearly the binding site of the aldehyde moiety in relation to the other substrate dihydroxyacetone phosphate.

Enzyme mediated C-C bond formation.

A number of significant biocatalysts have been investigated over the past year to improve accessibility, functional knowledge and use in developing synthetic applications. In particular, accumulating protein structural information has facilitated major advances in the detailed understanding of catalytic events and has, therefore, set the stage for rational protein engineering. This will have important implications for the future scope of practical applications, the first of which are currently being industrialized.

Practical synthesis of 4-hydroxy-3-oxobutylphosphonic acid and its evaluation as a bio-isosteric substrate of DHAP aldolase.

An efficient four step synthesis of the title compound 4-hydroxy-3-oxobutylphosphonate (2) has been developed based on inexpensive 4-ethoxy-1-hydroxybutane-2-one using an Arbusow reaction (59% overall yield). Several dihydroxyacetone-dependent aldolases having different stereospecificities were tested for their acceptance of this phosphonomethyl substrate mimic as the aldol donor. Individual enzymes belonging to both type I (Schiff base formation) and type II (Zn2+ catalysis) mechanistic classes were found to catalyze the stereoselective addition of 2 to simple aldehydes to provide bio-isosteric analogs of sugar 1-phosphates in high yields. The lack of acceptance by specific enzymes is discussed with regard to recent protein X-ray data.

Quo vadis photorespiration: a tale of two aldolases.

An O2-consuming side reaction of D-ribulose 1,5-bisphosphate carboxylase causes photorespiration in plants. This reaction may be an inevitable consequence of the enzyme's inability to protect its ene-diolate reaction intermediate from O2, a notion that is supported by the failure of persistent efforts to eliminate selectively its oxygenase activity by genetic manipulation. We have examined two a1dolases with similar ene-diolate intermediates, L-rhamnulose 1-phosphate aldolase and L-fuculose 1-phosphate aldolase. The former enzyme has an oxygenase activity, while the latter does not, suggesting that the reaction with O2 is not inevitable.

Activation of a cryptic gene encoding a kinase for L-xylulose opens a new pathway for the utilization of L-lyxose by Escherichia coli.

A silent gene encoding a kinase that specifically phosphorylates L-xylulose was activated and rendered constitutive in mutant cells of Escherichia coli. L-Xylulose kinase was purified to homogeneity and found to be a dimer of two subunits of 55 kDa, highly specific for L-xylulose with a Km of 0.8 mM, a Vmax of 33 mumol/min/mg, and an optimum pH of 8.4. Physical (thin layer chromatography) and spectroscopic (nuclear magnetic resonance and optical rotation) characterization of the product of L-xylulose kinase indicated that the enzyme phosphorylated the sugar at position 5. The gene encoding L-xylulose kinase was mapped in the 80.2 min region of the chromosome by conjugation and transduction. Cloning and comparison of the restriction map with the Kohara map (Kohara, Y., Akiyame, K., and Isono, K. (1987) Cell 50, 495-501) located the gene between positions 3963 and 3965 kilobases. The molecular and functional features of L-xylulose kinase together with the location of the corresponding gene indicate that this enzyme did not derive from mutation of any other known kinase. The new kinase opens a route for the utilization of L-lyxose through the action of rhamnose permease, rhamnose isomerase, and the phosphorylation of the L-xylulose formed to L-xylulose 5-phosphate, which is then introduced into the pentose phosphate pathway for subsequent metabolism.

Phosphoenolpyruvate as a dual purpose reagent for integrated nucleotide/nicotinamide cofactor recycling.

An efficient technique is presented which integrates cofactor dependent enzymic phosphorylation and dehydrogenation into a single, closed-loop system by employing phosphoenolpyruvate as the sacrificial reagent for sequential ATP and NAD+ recycling steps. Exemplary applications are developed for the synthesis of 6-phosphogluconate from glucose, and that of dihydroxyacetone phosphate from glycerol. The latter system is combined with exergonic diastereoselective aldol additions for the one-flask synthesis of a ketosugar (D-sorbose), thiosugar (L-threo-5-thiopentulose), or a sugar acid (L-threo-pent-4-ulosonic acid) starting from a mixture of glycerol and simple aldehydes.

L-lyxose metabolism employs the L-rhamnose pathway in mutant cells of Escherichia coli adapted to grow on L-lyxose.

Escherichia coli cannot grow on L-lyxose, a pentose analog of the 6-deoxyhexose L-rhamnose, which supports the growth of this and other enteric bacteria. L-Rhamnose is metabolized in E. coli by a system that consists of a rhamnose permease, rhamnose isomerase, rhamnulose kinase, and rhamnulose-1-phosphate aldolase, which yields the degradation products dihydroxyacetone phosphate and L-lactaldehyde. This aldehyde is oxidized to L-lactate by lactaldehyde dehydrogenase. All enzymes of the rhamnose system were found to be inducible not only by L-rhamnose but also by L-lyxose. L-Lyxose competed with L-rhamnose for the rhamnose transport system, and purified rhamnose isomerase catalyzed the conversion of L-lyxose into L-xylulose. However, rhamnulose kinase did not phosphorylate L-xylulose sufficiently to support the growth of wild-type E. coli on L-lyxose. Mutants able to grow on L-lyxose were analyzed and found to have a mutated rhamnulose kinase which phosphorylated L-xylulose as efficiently as the wild-type enzyme phosphorylated L-rhamnulose. Thus, the mutated kinase, mapped in the rha locus, enabled the growth of the mutant cells on L-lyxose. The glycolaldehyde generated in the cleavage of L-xylulose 1-phosphate by the rhamnulose-1-phosphate aldolase was oxidized by lactaldehyde dehydrogenase to glycolate, a compound normally utilized by E. coli.