Gjalt Huisman

Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture

Biocatalytic Boost Enzymes tend to direct reactions toward specific products much more selectively than synthetic catalysts. Unfortunately, this selectivity has evolved for cellular purposes and may not promote the sorts of reactions chemists are seeking to enhance (see the Perspective by Lutz). Siegel et al. (p. 309) now describe the design of enzymes that catalyze the bimolecular Diels-Alder reaction, a carbon-carbon bond formation reaction that is central to organic synthesis but unknown in natural metabolism. The enzymes display high stereoselectivity and substrate specificity, and an x-ray structure of the most active enzyme confirms that the structure matches the design. Savile et al. (p. 305, published online 17 June) applied a directed evolution approach to modify an existing transaminase enzyme so that it recognized a complex ketone in place of its smaller native substrate, and could tolerate the high temperature and organic cosolvent necessary to dissolve this ketone. This biocatalytic reaction improved the production efficiency of a drug that treats diabetes. An engineered enzyme offers substantial efficiency advantages in the production-scale synthesis of a drug. Pharmaceutical synthesis can benefit greatly from the selectivity gains associated with enzymatic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asymmetric enamine hydrogenation for the large-scale manufacture of the antidiabetic compound sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chemistry but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufacturing setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resolution. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manufacture of pharmaceuticals.

The dps promoter is activated by OxyR during growth and by IHF and σs in stationary phase

Dps is a non‐specific DNA‐binding protein abundant In starved Escherichia coli cells and is important for the defence against hydrogen peroxide. We found that dps mRNA levels are controlled by rpoS‐encoded σs, the transcriptional activator OxyR and the histone‐like IHF protein, in exponentially growing cells, dps is induced by treatment with hydrogen peroxide in an OxyR‐dependent manner. This OxyR‐dependent induction occurs only during log phase, although the OxyR protein is present in stationary phase, in the stationary phase cells, dps is expressed in a σs ‐ and IHF‐dependent manner. The purified OxyR and IHF proteins are also shown to bind upstream of the dps promoter. Our results suggest that the dps promoter is recognized by both σ70‐holoenzyme and σs‐holoenzyme, since OxyR acts through σ70 and the starts of the OxyR‐ and σs‐dependent transcripts are identical.

A functionally diverse enzyme superfamily that abstracts the alpha protons of carboxylic acids

Mandelate racemase and muconate lactonizing enzyme are structurally homologous but catalyze different reactions, each initiated by proton abstraction from carbon. The structural similarity to mandelate racemase of a previously unidentified gene product was used to deduce its function as a galactonate dehydratase. In this enzyme superfamily that has evolved to catalyze proton abstraction from carbon, three variations of homologous active site architectures are now represented: lysine and histidine bases in the active site of mandelate racemase, only a lysine base in the active site of muconate lactonizing enzyme, and only a histidine base in the active site of galactonate dehydratase. This discovery supports the hypothesis that new enzymatic activities evolve by recruitment of a protein catalyzing the same type of chemical reaction.