Fused dimerization increases expression,solubility, and activity of bacterial dehydratase enzymes |
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| Authors: | Carlos Rullán‐Lind Ruth B. Pietri Melvin Vázquez‐Cintrón Abel Baerga‐Ortiz |
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| Affiliation: | 1. Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico;2. Molecular Sciences Research Center, University of Puerto Rico, San Juan, Puerto Rico;3. Department of Chemistry, University of Puerto Rico, Cayey Campus, Cayey, Puerto Rico |
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| Abstract: | FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ‐FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids. |
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| Keywords: | FabA FabZ fused dimerization fatty acids enzyme engineering peptide linkers |
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