Yoshikatsu Kanai

In Thermus thermophilus, an aerobic Gram‐negative eubacterium used as a model organism, more than half of the phosphorylation sites identified by proteomic analysis are located near the ligand‐binding site, including the active site, of the enzyme in the three‐dimensional structure. We investigated the effect of these phosphorylation events on the activity of six enzymes (three nucleoside monophosphate kinases, isocitrate kinase, malate dehydrogenase and inorganic pyrophosphatase) by introducing phosphomimetic mutations, Glu, into the phosphorylation sites. All phosphomimetic mutants showed severely reduced activity compared with the wild‐type, particularly in the turnover number. The proteins analyzed in this study belong to different families and have various functions. This suggests that there is a widespread mechanism by which phosphorylation of amino acid residues near the active site reduces enzyme activity independent of the protein family and function.

Abstract Zinc finger domains are important interaction modules for binding to nucleic acids, proteins, lipids and small molecules. Many small-sized zinc finger proteins are encoded in bacterial genomes, but most of them have not been functionally annotated. We focused on TTHA0897, ZifS, as a small zinc finger protein from the extremely thermophilic eubacterium Thermus thermophilus HB8. In vivo experiments suggested that the cellular function of ZifS is related to the growth transition of T. thermophilus from the lag to the exponential phase under nutritionally limited conditions. In vitro biochemical experiments, including electrophoretic mobility shift assay and pull-down assay, yielded no clues about molecular functions of ZifS. X-ray crystallographic analysis revealed that the dimeric ZifS globally forms a cylinder-like structure, although ZifS dimer has no overall structural similarity to other known zinc finger proteins. The zinc ion-binding manner of ZifS fitted the characteristics of the zinc ribbon fold, which are mostly found in domains from proteins involved in the transcriptional and translational machinery. The crystal structure of ZifS is the first experimental insight into the molecular structure of this protein family, revealing several conserved features that may be functionally relevant.