Time-resolved structures of hydroxymethylbilane synthase (Lys59Gln mutant) as it is loaded with substrate in the crystal determined by Laue diffraction

Abstract

The structure of the catalytically active, reduced, form of the enzyme hydroxymethylbilane synthase (HMBS, Lys59Gln mutant) has been studied by Laue diffraction as the substrate, porphobilinogen (PBG), was fed to an immobilised crystal in a flow cell. Laue data at short time-scale time points (i.e. 1, 2, 4 and 8 min) were measured using several crystals and then averaged. Longer time-point data sets (i.e. 25 min ± 7 min and 2 h 23 min ± 9 min) were measured from individual crystals. All data sets benefited from using rapid exposures on ESRF ID09 (≈1 ms) and the fast duty cycle ESRF II CCD (readout time 8s). In one case, the substrate supply to a crystal in the flow cell was stopped at 4 h and monochromatic data collected at 12 h ± 30 min on ESRF BM14 (i.e. about 8 h after the substrate supply was stopped). Structural analysis of these data sets at all the time points was undertaken commencing with rigid-body refinement based upon the molecular model of the active, reduced, enzyme. The rigid-body refinement showed that rotational and translational movements of individual domains of the protein are less than 0.7° and 0.2 Å, respectively in the crystal with respect to the wild-type active form model. Moreover, difference Fourier maps at different time points versus the wild-type reduced form were calculated based upon the calculated phases from the wild-type reduced form model. These maps show that at 8 min, 25 min and 2 h, extended electron density appears in the active site region. This electron density is not visible in the 12 h case. Detailed structural refinement on the 2 h data, for which the extra electron density is most prominent, allowed an improved omit-type difference map to be calculated. This shows electron density in the active site adjacent and above the side-chain of Asp84, which plays a pivotal role throughout the catalytic reaction cycle. The density peak commences at the cofactor C2 ring (oxidised form) position (earlier proposed as a binding site for PBG). It then extends up towards Arg149, past Arg155 (residues whose mutation causes build up of ES₁ and ES₄ intermediate enzyme–substrate complexes, respectively) and out towards the open solvent channel of the crystal.