The role of the conserved histidine-aspartate pair in the ‘base-off’ binding of cobalamins

Abstract The conserved cobalamin-binding domain of glutamate mutase exists as a separate dissociable subunit, MutS. The results obtained from BIAcore analysis indicate that MutS alone, in the absence of E component of glutamate mutase (MutE, catalytic subunit), is capable of binding hydroxocobinamide (OHCbi) with a K d of 15.4±1.6 μM, but fails to bind adenosylcobalamin (AdoCbl). The UV–visible spectrum indicates that histidine ligation to the cobalt atom only occurs when both MutE and MutS are present in the solution. MutS mutants, MutS-D14N and MutS-H16G, are also capable of binding OHCbi, but their binding kinetics altered. Our experimental results show that the electrostatic interaction between histidine-aspartate pair is important in the binding of OHCbi or AdoCbl, no matter whether histidine coordinates to the cobalt atom or not. The catalytic subunit is also involved in histidine ligation to the cobalt atom. Meanwhile, mutation of either His16 or Asp14 significantly impairs the enzyme to cleave the cobalt–carbon bond of AdoCbl.