Alteration of the Flexible Loop in 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase Boosts Enthalpy-Driven Inhibition by Fosmidomycin

Abstract

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy–enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin’s phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.

Potent, specific MEPicides for treatment of zoonotic staphylococci

“…Comparison of the S. schleiferi DXR apoenzyme and FSM complex structures reveals how the C-terminal capping region (α9-α11 and α16-18) shift position to allow for the α10-α11 loop to position Trp196 adjacent to the inhibitor (Fig 3D). Movement of this flexible loop is a key feature for FSM inhibition of DXR from a variety of microorganisms(38). The residues that interact with FSM in the S. schleiferi DXR are conserved in the crystal structures of DXR from E. coli, P. falciparum , and M. tuberculosis with some variation in the sequence of the α10-α11 loop, although the tryptophan that contacts FSM is conserved in all these enzymes(34,36,37).…”

Targeting an Aromatic Hotspot in Plasmodium falciparum 1‐Deoxy‐d‐xylulose‐5‐phosphate Reductoisomerase with β‐Arylpropyl Analogues of Fosmidomycin

“…[16][17][18][19][20] Murkin and co-workersd emonstrated ac hange in the rate-limiting step of the Mycobacterium tuberculosis Dxr-catalyzed reaction upon alterationo fT rp203 in the flap, thereby establishing af unctional link between this amino acid and chemical barrier crossing. [21] Inhibition andb inding studies with fosmidomycin confirmed the importance of the flap, andt he conserved tryptophan in particular, for ligand binding. Structural evaluation of as eries of Dxr-bound compounds including 3 and 4 ( Figure 2) showed that the indole group of Trp211i nt he Escherichia coli enzyme (EcDxr) is displaced in order to accommodate the inhibitors' pyridine/quinoline rings, which form p-p stacking or chargetransfer interactions with the indole of the tryptophan side chain.…”

“…This flap is considered essential for the catalytic activity of Dxr . Murkin and co‐workers demonstrated a change in the rate‐limiting step of the Mycobacterium tuberculosis Dxr‐catalyzed reaction upon alteration of Trp203 in the flap, thereby establishing a functional link between this amino acid and chemical barrier crossing . Inhibition and binding studies with fosmidomycin confirmed the importance of the flap, and the conserved tryptophan in particular, for ligand binding.…”

Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

“…Furthermore, W203F and W203Y exhibited greatly diminished burst kinetics and larger primary 2 H KIEs on k cat than the wild-type enzyme (recall Section 3.2), indicating that mutation had rendered chemical steps more rate limiting. Surprisingly, fosmidomycin was found to bind more tightly to these mutants by up to an order of magnitude, a result that was attributable to a large increase in binding enthalpy [110].…”

“…Attempts to substitute this tryptophan with non-aromatic amino acids (Leu, Val or Ala) led to the mutants strongly impaired in both binding and catalytic properties [109]. Similarly, Kholodar and co-workers [110] demonstrated a lack of activity for W203G and W203A mutants of M. tuberculosis DXR. Interestingly, however, phenylalanine and tyrosine substitutions were well accommodated (Table 4), underscoring the necessity for an aromatic residue.…”