A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Small organic molecules, like phenylalanine and theophylline, are effective inhibitors of mammalian alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIAP). However, organic compounds do not hamper E. coli alkaline phosphatase (EcAP) activity. Sequence and structural analysis of alkaline phosphatase isozymes revealed a lack of conservation at EcAP residues that may be important for organic inhibition, thereby providing a potential explanation for the contrary inhibition. By mutating these EcAP residues to mimic analogous residues in mammalian APases, uncompetitive inhibition of EcAP by a class of aromatic molecules was conferred. While variants with single mutations are unaffected by organic effectors, variants expressing multiple mutations are inhibited, suggesting a synergistic relationship essential for organic binding. Circular dichroism was utilized to verify similar stability and kinetics for each variant. Michaelis‐Menten experiments were used to identify inhibition and confirm similar pH and cofactor dependence. The importance of key residues was determined based on their ability to confer organic inhibition. Furthermore, utilization of a set of purine derivatives allowed determination of the structure‐activity relationship for inhibitor binding. Specifically, this analysis enabled identification of hydrogen bonding requirements for organic inhibition. In total, designed substitutions altering EcAP residues near the active site to mimic analogous residues in mammalian APases confers uncompetitive inhibition by specific derivatives of a class of aromatic organic molecules.Support or Funding InformationResearch generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity grant (MRM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.