Further Insight into Crystal Structures of Escherichia coli IspH/LytB in Complex with Two Potent Inhibitors of the MEP Pathway: A Starting Point for Rational Design of New Antimicrobials

Abstract

IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7 Å resolution, respectively. The structure of the E. coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.

Isoprenoid Biosynthesis in Pathogenic Bacteria: Nuclear Resonance Vibrational Spectroscopy Provides Insight into the Unusual [4Fe‐4S] Cluster of the E. coli LytB/IspH Protein

“…3 contains the results of calculated NRVS data based on X-ray structure data. [9, 10, 20] There is reasonable agreement between calculated and experimental data and we attribute the deviations to the presence of slightly different protein conformations in protein crystals and in solution. For LytB in complex with 4 a NRVS data set has been calculated based on the structure recently published by the groups of Groll and Oldfield (see Figure 4Sc in the supplement), which for no obvious reasons, show significantly less agreement with the experimental data displayed in Fig.…”

“…Simulated NRVS data obtained via combined quantum chemical and molecular mechanics (QM/MM) calculations based on crystal structures of the active site/inhibitor complexes: (a) LytB with substrate 3 (3KE8.pdb), [9] (b) LytB in complex with inhibitor 4 (3ZGL.pdb), [20] and (c) LytB in complex with inhibitor 5 (4H4E.pdb). [10] …”

Methylerythritol Phosphate Pathway: Enzymatic Evidence for a Rotation in the LytB/IspH‐Catalyzed Reaction

“…coli IspH was produced, purified under anaerobic conditions and characterized as described previously. [21,29] The UV/Vis spectrum, the iron and sulfur content and the activity (750 nmol min À1 mg À1 )w ere in agreement with previously reported data [8,21,29] confirming that the [4Fe-4S] cluster was intact.…”

“…[20] However this conformation could not be observed in another independently reported structure of this complex (PDB ID:3ZGL) in which the occupancy of the [4Fe-4S]w as verified to be 100 %. [21] IspH contains av ery oxygen sensitive[ 4Fe-4S] center that can dissociate (loss of the apical iron) during the crystallization process. Therefore, it cannotb ee xcludedt hat the alternative conformation observed in someX -ray structures was due to the loss of this iron.…”

“…A similar alternative conformation of ( E )‐4‐amino‐3‐methylbut‐2‐en‐1‐yl diphosphate (AMBPP), a HMBPP analogue in which the OH group was replaced by an amino group, was also reported in a IspH:AMBPP complex structure (PDB ID:4H4D) . However this conformation could not be observed in another independently reported structure of this complex (PDB ID:3ZGL) in which the occupancy of the [4Fe‐4S] was verified to be 100 % . IspH contains a very oxygen sensitive [4Fe‐4S] center that can dissociate (loss of the apical iron) during the crystallization process.…”