The Role of Dimerization by Escherichia coli HypB in Hydrogenase Biosynthesis

• Main Author: Cai, Fang
• Other Authors: Zamble, Deborah
• Language: en_ca
• Published: 2010
• Subjects: Escherichia coli; HypB; Dimerization; Hydrogenase; 0487
• Online Access: http://hdl.handle.net/1807/25441

Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.