Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

Abstract

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Bacteriophage protein PEIP is a potent Bacillus subtilis enolase inhibitor

“…SF2312 produced by the Micromonospora is a natural antibiotic against a range of bacteria under anaerobic conditions and a potent inhibitor for human enolases (Leonard et al, 2016). SF2312 and its derivative POMSF inhibit the enzymatic activity of human enolase 2 (ENO2) by occupying its catalytic site with low nanomolar affinity (3.4 nM) and competing with substrate (Krucinska et al, 2019). In contrast, PEIP has lower affinity for enolase and achieves allosteric inhibition by interacting with a major intermolecular interface of the octameric bacterial enolase and inducing structural rearrangements in loop regions surrounding the active site.…”

Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation

“…Therefore, K 239 and its surrounding region seem a hot spot for adverse reactions derived from antibody recognition. Among the several enolase inhibitors available (Scatena et al, 2008;Spring and Wold 1971;Chan et al, 2016;de A S Navarro et al, 2007;Jung et al, 2013), the natural antibiotic SF2312 that mimics the carbanion intermediate of enolase reaction (Krucinska et al, 2019) and the transition state analogue phosphonoacetohydroxamic acid (PhAH) (Muller et al, 2012;Capello et al, 2016) inhibit cancer cell proliferation. However, only PhAH has been used as a therapeutic drug, although its utility to treat allergic reactions is unlikely, since the role of αenolase in these outcomes seems independent of enzyme activity.…”

Regulation of Chlamydia RsbU phosphatase activity by the enolase product, phosphoenolpyruvate

“…Enolase may be developmentally regulated in Chlamydia because phosphorylated enolase has been detected in EBs but not in RBs in Chlamydia caviae (Fisher et al, 2015). Other potential regulators of enolase activity include inhibitors, such as fluoride, SF2312 phosphonate, and tropolone derivatives (Krucinska et al, 2019a; Krucinska et al, 2019b; Spring and Wold, 1971) or post-translational modification by lysine acetylation (Nakayasu et al, 2017).…”