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Introduction

In this study, we present high-resolution crystal structures of ThiL from the pathogen Acinetobacter baumannii (AbThiL) bound to substrate Thiamine monophosphate (TMP) and substrate analog adenylyl-imidodiphosphate (AMPPNP), and bound to its products TPP and adenosine diphosphate (ADP). The structures highlight both global structural differences and differences regarding ligand binding between AbThiL and comparable structures from Aquifex aeolicus (AaThiL). Anomalous diffraction could also confirm a plasticity of AbThiL with regards to metal identity in the active site. The structures further support proposals made on the reaction mechanism for AaThiL.

Crystallization

Crystals grown in the presence of 5 mM AMPPNP/TMP/MgCl2 had many growth defects. Hence, co-crystals of AbThiL with 5 mM AMPPNP/MgCl2 from an optimization screen based on MCSG1 G4 (19% w/v PEG 3350, 200 mM K/Na-tartrate, 100 mM HEPES/NaOH pH 7.5) were soaked overnight with 5 mM AMPPNP/TMP/MgCl2. Crystals were harvested in two steps with reservoir with 20% v/v EG, 5 mM AMPPNP/TMP/MgCl2 added as the cryo protectant.