Calcium Chloride Enhance Crystal Growth of an Uncharacterized GNAT Enzyme

Abstract

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 InformationResearch reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).