Introduction

Alcohol dehydrogenases catalyze the interconversion of alcohols and the corresponding carbonyl compounds (aldehydes or ketones). Because this type of redox reaction is often required in organic synthesis and because such transformations often involve chirality, dehydrogenases have been explored by many workers to develop their synthetic potential (Fig. 1). Early work with readily available alcohol dehydrogenases from bakers' yeast1 and horse liver2 provided the first demonstration that these enzymes possessed high stereoselectivities and somewhat broad substrate tolerance. Lack of easy access to a larger range of dehydrogenases was a major impediment to further growth, however. This led to a situation in which a great deal of information was available for a small number of dehydrogenases. Today, the situation is almost completely reversed. The explosion in genome sequencing has provided an avalanche of potentially useful new alcohol dehydro-genases in the form of putative open reading frames. Making best use of these new proteins will require changing the way that dehydrogenase studies are carried out, and suggestions for how best to use these new resources, along with advances in other areas, are the subject of this chapter.

Regardless of the specific end-use, biocatalytic studies can be roughly divided into two phases: discovery and development. The major goal of the discovery phase is identifying a suitable enzyme that solves the chemical problem, in this

Figure 1: General dehydrogenase mechanism. In this example, the A hydride of NAD(P)H is transferred to the carbonyl substrate, which is activated by interaction with a Lewis acid (LA). A proton is donated to the developing oxyanion by a general acid (HX).

case, alcohol oxidation or carbonyl reduction. This search may involve interrogating a collection of naturally occurring dehydrogenases, a library of mutated proteins or some combination of the two. Success is defined by finding an enzyme with an acceptable combination of environmental tolerance, kinetic properties and stereoselectivity. Speed is often of the essence, particularly for applications in the pharmaceutical arena where time-to-market pressures dominate. Finding the "right" enzyme is only part of the solution, however. Process conditions must then be developed that yield the desired product at an acceptable rate, concentration and purity level. Often, what appeared to be an acceptable enzyme in the discovery phase must be improved or replaced during process development. Economic considerations also become important during the process development phase, since a biocatalytic solution is often compared with other synthetic strategies (transition metal catalysts, chiral pool starting materials, etc.) and the bioprocess must make economic sense if it is to be the final choice.

The fact that relatively few commercial processes currently utilize dehydro-genases,3 in spite of the high level of interest at the research and bench scales, argues that progress is needed in this area before these enzymes can be considered to be a normal part of chemical synthesis. Many of these advances will come by partnerships with genomics, nanotechnology and computational approaches. In the discovery area, key questions for the future are:

• Can dehydrogenase properties be predicted reliably from primary sequence data alone?

• Can dehydrogenase substrate- and stereoselectivities be altered predictably?

• Can the time and cost required to screen novel dehydrogenases be decreased significantly?

• Will it be possible to use individual dehydrogenase modules from large assemblies such as polyketide synthases?

• Can dehydrogenase active sites catalyze other types of 1,2-carbonyl additions?

Additional questions for the process development area include:

• Can the kinetic properties of dehydrogenases be improved?

• How can dehydrogenases catalyze reactions of very hydrophobic substrates?

• Can "cofactorless" dehydrogenases be developed that eliminate the need for nicotinamides?

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