The ROK kinase N-acetylglucosamine kinase uses a sequential random enzyme mechanism with successive conformational changes upon each substrate binding

N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolize environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase N-acetylglu-cosamine kinase (NagK) performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Ple-siomonas shigelloides in complex with GlcNAc and the ATP analog AMP-PNP. Surprisingly, PsNagK showed distinct conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Our molecular dynamics modeling of catalytic ion binding confirmed the location of the essential catalytic metal. Addi-tionally, site-directed mutagenesis confirmed the catalytic base and that the metal-coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.

Automated summary

N-acetyl-D-glucosamine (GlcNAc) is an important component in bacterial cell walls and its metabolism starts with phosphorylation to GlcNAc-6-phosphate. The ROK kinase NagK is responsible for this step in bacteria. In this study, the structure of NagK in complex with GlcNAc and ATP analog AMP-PNP was solved, revealing conformational changes and a sequential random enzyme mechanism. Molecular dynamics modeling and site-directed mutagenesis further confirmed the essential catalytic metal and base residues. Overall, this study provides comprehensive insights into the activity of ROK kinases.