Metal-dependent nucleotide binding to the Escherichia coli rotamase SlyD

Upon expression and purification of the first catalytic domain of mammalian adenylate cyclase type 1 (IC1), a 27 kDa contaminant was observed, which w as labelled by three radioactive ATP analogues (8-azido-ATP, 3'-O-(4-benzoy l)benzoyl-ATP and 2',3'-dialdehyde-ATP); the protein was purified separatel y and identified as Escherichia coli SlyD by N-terminal amino acid sequence determination. SlyD is the host protein required for lysis of E. coli upon infection with bacteriophage Phi X174 and has recently been shown to displ ay rotamase (peptidylproline cis-trans-isomerase) activity. The covalent in corporation of ATP analogues into SlyD was promoted by bivalent transition metal ions (Zn2+ greater than or equal to Ni2+ > Co2+ > Cu2+) but not by Mg 2+ or Ca2+, this is consistent with the known metal ion specificity of SlyD , ATP, ADP, GTP and UTP suppressed labelling of SlyD with comparable potenc ies. Similarly, SlyD bound 2',3'-O-(-2,4,6-trinitrophenol)-ATP with an affi nity in the range of 10 mu M, as determined by fluorescence enhancement. Th is interaction was further augmented in the presence of Zn2+ (K-d = similar to 2 mu M at saturating Zn2+) but not of Mg2+. Irrespective of the assay c onditions, hydrolysis of nucleotides by SlyD was not detected. Upon gel fil tration on a Superose HR12 column, SlyD (predicted molecular mass = 21 kDa) migrated with an apparent molecular mass of 44 kDa, indicating that the pr otein was a dimer. However, the migration of SlyD was not affected by the p resence of Zn2+ or of Zn2+ and ATP. Thus we concluded that SlyD binds nucle otides in the presence of metal ions. these findings suggest that SlyD serv es a physiological role that goes beyond that accounted for by its intrinsi c rotamase activity, which is observed in the absence of metal ions.