Dihydrofolate Reductase

This chapter begins with an overview of the synergy between rational design and screening or selection. The choice of a rational design approach requires consideration of the number of enzyme variants that can be evaluated experimentally. A review of fundamentals of enzyme catalysis from an energy landscape point of view is presented to illustrate why and where rational design could be applied. The bulk of the chapter follows with many examples from the literature of techniques that have worked. Throughout the discussion, mention of basic computational techniques needed to support design is made. It will be seen that it is often quite feasible to adapt an enzyme to many chemical process or organism-engineering needs. Catalytic effects that do not fall well into a thermodynamic viewpoint, such as vibrations or tunneling, are generally smaller, but could also be treated computationally with more development effort. Mutations to proline place a kink in the backbone that can also be stabilizing. The procedure of finding proline mutations can be automated by use of side-chain repacking software. The use of similar sequences to suggest mutations during the evolution of an enzyme is one of the more effective and easiest approaches to stabilization of proteins. Mutations at four positions designed from direct structure comparison helped convert an isocitrate dehydrogenase to an isopropylmalate dehydrogenase, although it was subsequently shown that random mutagenesis was more effective.

Cold-adapted enzymes are produced by microorganisms living at permanently low temperature, which constitutes the major environment on planet Earth and includes deep sea, polar, and mountain regions. This chapter deals with those enzymes that are significantly adapted to low temperatures, that is, displaying a high specific activity at low temperatures. Many enzymes produced by cold-adapted microorganisms have now been fully characterized in terms of their physical, chemical, and kinetic properties but still only 11 structures have been solved by X-ray crystallography: α-amylase, citrate synthase, malate dehydrogenase, triosephosphate isomerase, Ca2+-Zn2+ protease, xylanase, adenylate kinase, cellulase, subtilisin-like protease, tyrosine phosphatase, and β-galactosidase. The in vitro growth temperature of these psychrophilic microorganisms is very important for enzyme production, especially for extracellular enzymes, since the production is highly dependent on temperature. In another systematic investigation, the production of various extracellular enzymes such as cellulases, pectate lyases, chitinases, and chitobiases by several strains permanently or seasonally exposed to cold temperatures was followed as a function of growth temperature. The structural modifications believed to be involved in cold-adaptation have been examined in some limited cases using site-directed mutagenesis and directed evolution approaches. The two main properties of cold-adaptation enzymes—a high specific activity at low and moderate temperatures and a low thermostability enabling their rapid inactivation in a complex mixture—render these enzymes particularly suitable for various low to moderate temperature biotechnological processes.

This chapter focuses on the crystal structure of the RNA binding domain of BstL2, and discusses its structure from functional and evolutionary points of view. Site-directed mutagenesis of Arg86 or Arg155 significantly diminished RNA binding affinity, and in addition, Arg68 and Lys70 mutations caused partial loss of RNA binding. To date, the three-dimensional structures of over a dozen ribosomal proteins have been determined. Comparison of their structures with those of other known proteins in the Protein Data Bank revealed that many ribosomal proteins share structural motifs, such as RNP, dsRNA binding domain, KH domain, and helix-turn-helix motifs, with RNA or DNA binding proteins. Recent studies of Thermus aquaticus ribosomes, however, demonstrated that peptidyltransferase activity is never attributed solely to the 23S rRNA, and they reduce the number of possible essential macromolecular components of the peptidyltransferase center to 23S rRNA and ribosomal proteins L2 and L3.

The ability of aminoacyl-tRNA synthetases (aaRS) to faithfully recognize both their amino acid and tRNA substrates is essential for accurate protein synthesis. This chapter focuses on the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). This system is arguably the best characterized aaRS-tRNA interaction both functionally and structurally, as the first high-resolution crystal structure of a protein-RNA complex was solved for GlnRS:tRNAGln. The chapter summarizes the characteristics of the GlnRS:tRNAGln system that make GlnRS unique among aaRS, as well as those that make the glutamine system an ideal model for the study of protein-RNA interaction, specifically aaRS-tRNA interaction. Together the crystal structure, genetic, and biochemical studies have identified the most important specificity determinants from among the hundreds of contacts between GlnRS and tRNAGln. The chapter also describes several of the more interesting aspects of the GlnRS:tRNAGln system: (1) the close evolutionary relationship between GlnRS and glutamyl-tRNA synthetase (GluRS); (2) GlnRS's relaxed discrimination against noncognate tRNAs coupled with its "overdetermined," tight recognition of its cognate tRNA; and (3) the enzyme mechanism of GlnRS, specifically the structural and functional communication that permits this small monomeric aaRS to recognize tRNA identity elements that are more than 75Å apart in uncomplexed tRNA. The information obtained from biophysical techniques (crystallography, fluorescence, and x-ray/neutron scattering) and from genetic and biochemical approaches is combined to yield a coherent and detailed picture of the specific recognition of tRNA by E. coli GlnRS.