Mirror‐Image Packing Provides a Molecular Basis for the Nanomolar Equipotency of Enantiomers of an Experimental Herbicide

Abstract

Programs of drug discovery generally exploit one enantiomer of ac hiral compound for lead development following the principle that enantiomer recognition is central to biological specificity.However,chiral promiscuity has been identified for anumber of enzyme families,whichhave shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzymesactive site.Reported here is as eries of crystallographic studies of complexes between an enzyme and ap otent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with ar acemate at 1.85 resolution failed to identify the chirality of the bound inhibitor,h owever,b y extending the resolution to 1.1 and by analyzing highresolution complexes with the enantiopure compounds,w e determined that both enantiomers make equivalent pseudosymmetric interactions in the active site,t hus mimicking an achiral reaction intermediate.

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…Beyond racemases ande pimerases, there are examples of other enzymes that are capable of binding enantiomers, althougho ne enantiomer is typicallyt he substrate, while the other may be an inhibitor.Inmany cases, mirror-image packing has also been observed. [261,262] That these enzymes already possess the ability to bind enantiomersw ith the same orientations as the substrates of racemases and epimerases is intriguing since it suggests that these enzymes might be amenable to engineering of racemase or epimerase activity by rational designa nd/ord irected evolution with, of course, concomitant reduction or obviation of their native activity. [a] alaniner acemase (AlaR) 1L6F (PLP-l-Ala, 2.0 ) [69] 1L6G (PLP-d-Ala, 2.0 ) [69] 2VD9 (PLP-l-AlaPand PLP-d-Ala, 2.1 ) [71] 1.02 1.33 mirror isoleucine 2-epimerase (IleE)5WYA(PLP-l-isoleucine,2 .65 ) [74] 5WYF (PLP-d-allo-isoleucine, 2.12 ) [74] 0.75 mirror serine racemase (SerR) 2ZR8 (serine, 2.2 )model [81] 0.48 super/NS a-amino-e-caprolactam racemase (ACLR) PDB 5M49(l-a nd d-a-amino-e-caprolactam, 1.51 ) [83] 1.43 mirror/NS glutamate racemase( GluR) 2JFO (l-glutamate and d-glutamate, 2.5 ) [84] - [b] possibly super aspartater acemase (AspR) 5WXY (l-aspartate, 2.63 ) [85] 5WXZ (d-aspartate, 2.80 ) [85] 1.00 mirror Asp/Glu racemase 5HRC (l-aspartate, 1.76 ) [86] 5HRA (d-aspartate, 1.60 ) [86] 1.16 mirror diaminopimelate epimerase (DAPE) 2GKJ (d,l-aziridino-DAP, 1.55 ) [87] 2GKE (l,l-aziridino-DAP,1.70 ) [87] 3EKM (d,l-aziridino-DAP,1.95 ) [88] 3EJX (l,l-aziridino-DAP,2 .30 ) [88] 0.87 0.54 mirror [a] proliner acemase(ProR) 1W61 (pyrrole-2-carboxylate, 2.10 ) [136] -NS l-Ala-d/l-Glud ipeptide epimerase (AEE) 3R1Z (l-Ala-l-Glua nd l-Ala-d-Glu, 2.9 ) [89] 1.12 [b] mirror histidine racemase (HisR) model based on 5ZL6 (PLP,2 .10 ) [142] model based on 6JIS (apo, 1.82 ) [90] 1.67 [b] mirror a-methylacyl-coenzyme Aracemase (epimerase) (AMACR) 2GCE ((2S)-ibuprofenoyl-CoA and (2R)-ibuprofenoyl-CoA, 1.85 ) [91] 2GD0((2S)-methylmyristoyl-CoA, 1.70 ) [91] 2GCI ((2R)-methylmyrist...…”

The expanding toolkit for structural biology: synchrotrons, X-ray lasers and cryoEM

“…Another example is the case of imidazoleglycerolphosphate-dehydratase (IGPD), an essential enzyme in histidine biosynthesis. The Arabadopsis homologue was highly amenable to crystallization, resulting in several high-resolution structures (Bisson et al, 2016). These structures not only revealed the mode of binding, but also the molecular basis for the nanomolar equipotency of potent enantiomers.…”

Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM

“…IGPD catalyzes the sixth step in histidine biosynthesis, where manganese(II)-dependent dehydration of imidazoleglycerol-phosphate occurs to form imidazoleacetol-phosphate and a concomitant water ( 1 – 3 ). The structure of IGPD has been well studied by X-ray crystallography in several organisms, including the plant Arabidopsis thaliana ( At _IGPD) ( 3 ), the bacterial and archaeal species including Mycobacterium tuberculosis and Pyrococcus furiosus ( 4 , 5 ), and the fungus Cryptococcus neoformans ( 6 ). These studies show that the core IGPD structure is conserved, comprising a 24-mer with 432 symmetry with two octahedrally coordinated Mn 2+ ions in each active site.…”

“…Although there is generally high sequence homology between IGPD homologs from different species, especially surrounding the active site, Sc _IGPD has an intriguing sequence difference arising from a 24-amino acid insertion between β2 and β3 that is only found in budding yeast. Interestingly, the most potent lead compound that has emerged from herbicide development, the triazole-phosphonate compound 2-hydroxy-3-(1,2,4-triazol-1-yl) (C348), is an order of magnitude more potent against Sc _IGPD (0.6 nM K i ) ( 2 ) than At_ IGPD (∼25 nM K i ) ( 5 ). Although the mode of binding of C348 has been well studied by X-ray crystallography in At _IGPD, the molecular basis of this difference in potency is unknown.…”

“…Here we present single-particle cryo-EM structures of At _IGPD and Sc _IGPD in complex with C348 to global resolutions of 3.1 and 3.2 Å, respectively. The core of each complex displays higher local resolution, permitting de novo building and the identification of both the bound inhibitor and coordinating metal ions, with the At structure validated through a corresponding X-ray structure (PDB ID: 5EKW) ( 5 ). Importantly, clear structural differences could be seen between the At and Sc IGPD homologs that may account for the significant differences seen between the binding affinities of potent inhibitors.…”