In all domains of life, zinc is an essential transition metal required for survival and pathogenic bacteria are no exception. During host infection and under zinc-limiting conditions, the transcriptional metalloregulator protein Zn2+-uptake regulator (Zur) from Pseudomonas aeruginosa upregulates the expression of several Zn2+-import pathway genes such as the Zur-regulated metallophore (Zrm) zrmABCD operon, which is responsible for the biosynthesis and transport of the Zn2+-binding opine metallophore pseudopaline. Biosynthesis of pseudopaline in the cytoplasm is a two-step enzymatic reaction consisting of ZrmB, a nicotianamine synthase-like (NAS) enzyme with S-adenosyl-L-methionine (SAM)-dependent aminoalkyl transferase activity and ZrmC, an opine dehydrogenase enzyme. Given that the crystal structure of ZrmB is unknown and the critical role ZrmB plays in metallophore-mediated Zn2+ acquisition, the main aim of this study is to structurally characterise ZrmB using X-ray crystallography.
The crystal structures of ligand-free and product-bound ZmrB were determined at 2.53 Å and 2.43 Å, respectively, and shows that it belongs to the family of SAM-dependent aminoalkyltransferases. It is made up of two domains, an N-terminal four-helical bundle domain followed by a C-terminal Rossman-like fold domain. The two domains are interconnected by a long flexible and disordered loop (N-C linker), which becomes ordered and forms an a-helix during catalysis. Structural superposition with the Staphylococcus aureus NAS orthologue, CntL, shows that they are very similar, with the main differences being the residues present at the active site and provides an explanation for the specificity of ZrmB and CntL for the L-Histidine and D-Histidine substrate, respectively. Structural comparison with the archaeal Methanothermobacter thermautotrophicus NAS orthologue, MtNAS, and structural homology search using the DALI server shows that the N-terminal a-helical bundle domain is unique to the NAS family. Future directions include obtaining the crystal structures of substrate-bound ZrmB and further biophysical characterisations such as its oligomerisation state in solution.