The Skaggs Institute
for Chemical Biology

Scientific Report 2008

Pathway Engineering for Enzymatic Synthesis

J.R. Williamson, W. Anderson, F. Agnelli,A. Beck, C. Beuck, A. Bunner, A. Carmel, S. Chen, S. Edgcomb, D. Kerkow, S. Kwan, E. Menicelli, W. Ridgeway, G. Ring, H. Schultheisz, L.G. Scott, Z. Shajani, E. Sperling, M. Sykes, B. Szymczyna, J. Wu

Enzymes are the protein factors in cells that are responsible for effecting chemical transformations of metabolites and macromolecules. Enzymes are responsible for synthesizing the thousands of small molecules in a cell that are necessary for a functioning metabolism and for synthesizing diverse natural products that can have important medicinal properties.

Enzymatic synthesis in the laboratory is a powerful alternative to organic chemical synthesis for certain types of molecules. Because enzymes have complex 3-dimensional folds, they can bind specifically to substrates and catalyze complex chemical reactions that are sometimes difficult to achieve with organic synthesis. A series of enzymes can be used simultaneously to effect a series of chemical reactions without isolation of the intermediate products. Thus, enzymatic synthesis is a powerful tool that can be applied to certain synthetic problems.

We are interested in the structure of RNA molecules and RNA-protein complexes that are important for translation or regulation of protein expression. One of the structural biology tools we use is nuclear magnetic resonance (NMR) spectroscopy, which can be used to determine the structure of macromolecules in solution. Application of NMR requires the incorporation of the stable isotope labels ¹³C and ¹⁵N, which can be difficult with RNA molecules. One aspect of our research program is developing methods to incorporate these stable isotope labels into RNA molecules, to enable structural studies.

We have developed a flexible and powerful enzymatic synthesis of the purine nucleotides ATP and GTP. The method is, to our knowledge, the most complex and intricately engineered enzymatic synthesis that has been carried out in a laboratory to date. The process requires 28 enzymes, each of which was overproduced in Escherichia coli and purified before synthesis. The longest linear series of reactions has 19 sequential steps, but the process can be carried out with a yield of about 60%. The scheme requires input of 3 types of reagents (Fig. 1). First, the starting material substrates are incorporated into the final nucleotide product. Second, trace amounts of catalytic cofactors, such as NAD, glutamine, aspartate, and tetrahydrofolate, are supplied. The cofactors are recycled by using metabolic enzymes. Third, excess amounts of fuel reagents such as creatine phosphate and α-ketoglutarate are added to drive the recycling reactions and to drive the overall synthesis from the starting materials to the products (ATP or GTP). Almost every one of the 19 key steps involves recycling of a cofactor and consumption of fuel molecules.

Fig. 1.One-pot de novo enzymatic synthesis of purine nucleotides. Glucose, carbon dioxide (CO2), ammonia (NH3), and serine, the stoichiometrically consumed reagents incorporated into products, are highlighted in yellow. A, Enzymes from the glycolytic pathway and purine biosynthetic pathways convert glucose into ATP and GTP. Intermediates are shown in the vertical sequence, and the abbreviation for each enzyme is shown in italics. Cofactors (e.g., ATP, NAD, THF) are shown to the left or the right of the main reaction sequence. The circular arrow symbols indicate the enzymatic regeneration of the cofactors, which are color coded for the reactions shown in the cofactor regeneration schemes (B). Red = ATP recycling, green = NAD recycling, purple = glutamine recycling, orange = aspartate recycling, blue = folate recycling.

The overall synthesis results in the efficient synthesis of ATP or GTP starting from glucose, carbon dioxide, ammonia, and serine. These precursors are available in isotopically labeled forms, and a variety of different labeling patterns can be constructed by using different combinations of the labeled precursors. Using this approach, we were able to synthesize isotopically labeled ATP and GTP with novel labeling patterns that have useful properties for NMR analysis of RNA structure.

Although preparing all of the enzymes for this biosynthetic approach is time-consuming, the resulting labeled nucleotides cannot easily be synthesized in any other way. The enzymatic synthesis approach we have developed should have broad applicability in the synthesis of other high-value biochemicals that can be used for structural biology or metabolic profiling experiments.

Publications

Edgcomb, S.P., Aschrafi, A., Kompfner, E., Williamson, J.R., Gerace, L., Hennig. M. Protein structure and oligomerization are important for the formation of export-competent HIV-1 Rev-RRE complexes. Protein Sci. 17:420, 2008.

Hennig, M., Scott, L.G., Sperling, E., Bermel, W., Williamson, J.R. Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J. Am. Chem. Soc. 129:14911, 2007.

Kiessling, L.L., Doudna, J.A., Johnsson, K., Mapp, A.K., Marletta, M.A., Seeberger, P.H., Williamson, J.R., Wedde, S.G. A higher degree of difficulty. ACS Chem. Biol. 2:197, 2007.

Naidoo, N., Harrop, S.J., Sobti, M., Haynes, P.A., Szymczyna, B.R., Williamson, J.R., Curmi, P.M.G., Mabbut, B.C. Crystal structure of Lsm3 octamer from Saccharomyces cerevisae: implications for Lsm ring organisation and recruitment. J. Mol. Biol. 377:1357, 2008.

Schultheisz, H.L., Szymczyna, B.R., Scott, L.G., Williamson, J.R. Pathway engineered de novo enzymatic purine nucleotide synthesis. ACS Chem. Biol. 3:499, 2008.

Sperling, E., Bunner, A., Sykes, M.T., Williamson, J.R. Quantitative analysis of isotope distributions in proteomic mass spectrometry using least-squares Fourier transform convolution. Anal. Chem. 80:4906, 2008.

Szymczyna, B.R., Gan, L., Johnson, J.E., Williamson, J.R. Solution NMR studies of the maturation intermediates of a 13 MDa viral capsid. J. Am. Chem. Soc. 129:7867, 2007.

Tahmassebi, D.C., Williamson, J.R. Balancing teaching and research in obtaining a faculty position at a predominantly undergraduate institution. ACS Chem. Biol. 2:521, 2007.

Williamson, J.R. Biophysical studies of bacterial ribosome assembly. Curr. Opin. Struct. Biol. 18:299, 2008.

Williamson, J.R. Cooperativity in macromolecular assembly. Nat. Chem. Biol. 4:458, 2008.