Bacillus anthracis is the causative agent of the deadly disease Anthrax. Its use in bioterrorism and its ability to re-emerge have brought renewed interest in this organism. B. anthracis is a Gram-positive bacterium that adds L-rhamnose to its cell-wall polysaccharides using the activated donor dTDP-β-L-rhamnose. The enzymes involved in the biosynthesis of the activated donor are absent in humans, which make them ideal targets for therapeutic development to combat pathogens. Here, the 2.65 Å resolution crystal structure of the fourth enzyme in the dTDP-β-L-rhamnose-biosynthetic pathway from B. anthracis, dTDP-4-dehydro-β-L-rhamnose reductase (RfbD), is presented in complex with NADP. This enzyme catalyzes the reduction of dTDP-4-dehydro-β-L-rhamnose to dTDP-β-L-rhamnose. Although the protein was co-crystallized in the presence of Mg, the protein lacks the conserved residues that coordinate Mg.

Our aim was to construct a library of 63 rationally designed aminoglycoside N‐acetyltransferase mutants in a high‐throughput manner using the Q5® site‐directed mutagenesis kit from New England BioLabs Inc. Per the manufacturer's recommendation, we used their NEBaseChanger™ tool to design primers for each mutant and followed their Quick Protocol. Since we wanted to use our 96‐well thermocycler with six different gradient optimization blocks, some of the recommended annealing temperatures were adjusted by ± 0.5°C. Qualitatively, we found that this change did not have a significant impact on obtaining amplified products for most of our samples. We observed 49 PCR products at the correct size of ~6 kB in our first reaction and proceeded with the subsequent steps of the manufacturer's protocol. One colony from each mutant was then sent for sequencing to save funds. Of the 49 constructs tested, 32 were correct, which resulted in a 65% success rate from a single reaction. We sent one additional colony from the remaining 17 constructs and obtained 7 additionally correct mutants, for a total of 39/49 correct constructs. The remaining 10 mutants from this first amplification showed products with premature stop codons, wild‐type template DNA, or incomplete amplification products. Therefore, we performed additional troubleshooting reactions to obtain the remaining 24/63 mutants. We found that adjustments of up to 3°C from the recommended annealing temperature and additional sequencing attempts were required to obtain the remaining mutants. While the Q5 kit is user‐friendly and straightforward, we found it was not as cost‐effective as we had hoped and required several rounds of troubleshooting to complete our library.Support or Funding InformationThis work was supported in part by a Ken Fong Fund Grant (to MLK and PP). Additional support was provided by the Genentech Foundation and NIH MBRS‐RISE: R25‐GM059298 (to OP).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.