Metal-dependent nucleotide binding to the Escherichia coli rotamase SlyD

• Published in: Biochemical journal 1999-08, Vol.342 (1), p.33-39
• Main Authors: MITTERAUER, Thomas, NANOFF, Christian, AHORN, Horst, FREISSMUTH, Michael, HOHENEGGER, Martin
• Format: Article
• Language: English
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3420033

Upon expression and purification of the first catalytic domain of mammalian adenylate cyclase type 1 (IC1), a 27 kDa contaminant was observed, which was labelled by three radioactive ATP analogues (8-azido-ATP, 3′-O-(4-benzoyl)benzoyl-ATP and 2′,3′-dialdehyde-ATP); the protein was purified separately and identified as Escherichiacoli SlyD by N-terminal amino acid sequence determination. SlyD is the host protein required for lysis of E.coli upon infection with bacteriophage ΦX174 and has recently been shown to display rotamase (peptidylproline cis-trans-isomerase) activity. The covalent incorporation of ATP analogues into SlyD was promoted by bivalent transition metal ions (Zn2+ ⩾ Ni2+ > Co2+ > Cu2+) but not by Mg2+ or Ca2+; this is consistent with the known metal ion specificity of SlyD. ATP, ADP, GTP and UTP suppressed labelling of SlyD with comparable potencies. Similarly, SlyD bound 2′,3′-O-(-2,4,6-trinitrophenyl)-ATP with an affinity in the range of 10 μM, as determined by fluorescence enhancement. This interaction was further augmented in the presence of Zn2+ (Kd = ∼ 2 μM at saturating Zn2+) but not of Mg2+. Irrespective of the assay conditions, hydrolysis of nucleotides by SlyD was not detected. Upon gel filtration on a Superose HR12 column, SlyD (predicted molecular mass = 21 kDa) migrated with an apparent molecular mass of 44 kDa, indicating that the protein was a dimer. However, the migration of SlyD was not affected by the presence of Zn2+ or of Zn2+ and ATP. Thus we concluded that SlyD binds nucleotides in the presence of metal ions. These findings suggest that SlyD serves a physiological role that goes beyond that accounted for by its intrinsic rotamase activity, which is observed in the absence of metal ions.