Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.

Gcn5‐related N‐acetyltransferases (GNAT) are widespread across all domains of life and are capable of acetylating an assortment of substrates. However, many of their structures and functions remain unknown in bacteria. Our laboratory currently studies uncharacterized GNATs from Pseudomonas aeruginosa. We previously found the enzyme PA3944 acetylated polymyxin antibiotics and observed there was a duplicate GNAT gene (PA3945) with approximately 75% identity on the same operon. This protein has not been structurally or functionally characterized. Therefore, we screened this enzyme for activity using a broad‐substrate screening assay and set up crystallization screens to obtain protein crystals. Unfortunately, the PA3945 enzyme did not acetylate any substrates in the kinetic screening assay, which indicates it has a separate role compared to the PA3944 enzyme. We identified crystallization conditions that yielded microcrystals in the presence of calcium chloride. We further optimized these conditions using calcium chloride as an additive, which yielded larger crystals. Currently, we are investigating whether calcium chloride plays a role in enzyme activity and whether we can further optimize the crystallization conditions to yield suitable crystals for X‐ray diffraction and structure elucidation. Support or Funding Information Research reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).