Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands

Abstract

Enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are attractive targets for the development of herbicides and drugs against infectious diseases. While this pathway is essential for many pathogens and plants, mammals do not depend on it for the synthesis of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway, is unique in that it has an allosteric regulatory site. We elucidated the binding mode of phenylisoxazoles, a new class of allosteric inhibitors. Allosteric inhibition is effected by large conformational changes of a loop region proximal to the active site. We investigated the different roles of residues in this loop by mutation studies and identified repulsive interactions with Asp291 and Asp292 to be responsible for inhibition. Crystallographic data and the response of mutant enzymes to three different classes of allosteric inhibitors provide an in-depth understanding of the allosteric mechanism. The obtained mutant enzymes show selective resistance to allosteric inhibitors and provide conceptually valuable information for future engineering of herbicide-resistant crops. We found that the isoprenoid precursors IPP and DMAPP are natural inhibitors of Arabidopsis thaliana IspD; however, they do not seem to bind to the allosteric site.

Thermodynamics, Kinetics and Mechanisms of Noncompetitive Allosteric Inhibition of Chymotrypsin by Dihydrolipoic Acid-Coated Gold Nanoclusters

“…The most common reversible regulation type is competitive inhibition, where inhibitors bind to the active sites of enzymes, blocking the access of substrates to enzymes. In addition, regulators can also bind to the allosteric sites, leading to the structural changes of enzymes. − There are many factors affecting enzyme regulation, such as sizes, shapes, and surface charges of both enzymes and regulators, as well as pH, temperature, and ionic strength of the environment. , Therefore, nanomaterials with tailored sizes and surface modifications are highly attractive as exogenous regulators. These characters allow us to design and synthesize different kinds of nanomaterials according to specific demands.…”

Synthesis and Evaluation of Halogenated 5-(2-Hydroxyphenyl)pyrazoles as Pseudilin Analogues Targeting the Enzyme IspD in the Methylerythritol Phosphate Pathway

“…Very recently, a phenylisoxazole compound, namely, 5-(3,5-dibromo-2-hydroxyphenyl)-3-trifluoromethyisoxazole (PIX), was also demonstrated to bind to the same allosteric site, with an IC 50 value of 9.3 μM (Figure ). It is worth mentioning that the presence of a divalent cation, such as Cd 2+ , could promote the binding of pseudilin with At IspD but had no effect on the binding of PIX …”

“…Nevertheless, some of the synthetic steps are valuable to the synthesis of IspD inhibitors with similar structural patterns. On the other hand, the synthesis of the phenylisoxazole PIX has not been reported . Unfortunately, although IspD is a promising target in developing new inhibitors, except the above-mentioned compounds, which were identified by high-throughput screening, the particular design and synthesis of potential compounds targeting IspD have not been reported thus far.…”

Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor

“…The DXP pathway is exclusively present in protozoan parasites from phylum Apicomplexa, such as malaria-causing Plasmodium spp., Toxoplasma gondii , and Cryptosporidium , as well as in plants, and in numerous pathogenic bacteria including Mycobacterium tuberculosis . Multidrug resistance remains a major obstacle to successful treatment of many human life-threatening and prevalent diseases including malaria, 18 , 23 , 24 new and previously treated cases of tuberculosis 25 – 27 and toxoplasmosis. 28 – 30 Apicomplexan parasites, such as Neospora , Babesia , Theilleria , as well as Toxoplasma , put a tremendous burden on food security through diseases in livestock and poultry.…”

“… 30 , 32 , 35 Moreover, these enzymes are clinically validated as drug targets in apicomplexans, pathogenic bacteria, and as targets for herbicides in plants. 19 – 21 , 23 , 24 , 36 – 39 Any potential inhibitor of the enzymes disrupting the metabolic cascade in the DXP pathway will be devoid of target-related toxicity. 40 , 41 …”