Feo is the most widely conserved system for ferrous iron transport in prokaryotes, and it is important for virulence in some pathogens. However, its mechanism of iron transport is not fully understood. In this study, we used full-length Vibrio cholerae FeoB (VcFeoB) as a model system to study whether its enzymatic activity is affected by regulatory factors commonly associated with FeoB proteins from other species or with G-proteins that have homology to FeoB. VcFeoB showed a higher rate of hydrolysis of both ATP and GTP than its N-terminal domain alone; likewise, ions such as K+ and Fe2+ did not modulate its nucleotide hydrolysis. We also showed that the three V. cholerae Feo proteins (FeoA, FeoB, and FeoC) work in a 1 : 1 : 1 molar ratio in vivo. Although both FeoA and FeoC are required for Feo-mediated iron transport, neither of these proteins affected the VcFeoB NTPase rate. These results are consistent with an active transport mechanism independent of stimulatory factors and highlight the importance of using full-length FeoB proteins as a reliable proxy to study Feo-mediated iron transport in vitro.
Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.