The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.
A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å . Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh -strands. This E101N/R230I mutant had an increased value of k cat /K m of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the -cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.The amidohydrolase superfamily of enzymes comprises members that catalyze hydrolytic reactions on a broad range of substrates bearing ester or amide functional groups with carbon or phosphorus centers (1). These reactions are mediated by a conserved mononuclear or binuclear center within a (/␣) 8 -barrel structural scaffold (2) and are initiated by the activation of a water molecule for nucleophilic attack on an activated scissile bond of the substrate for subsequent hydrolysis. The amidohydrolase superfamily was first described by Holm and Sander in 1997 (3) and has since expanded to cover more than 30 reactions involving a diverse range of substrates (2, 4), including quorum-sensing N-acylhomoserine lactones (AHLs) 4 (5). We recently reported the directed evolution of a member of the amidohydrolase superfamily that hydrolyzes AHLs (6); this quorum-quenching enzyme is part of a group of divergently related enzymes within the superfamily, the phosphotriesterase-like lactonases (PLLs), which hydrolyze quorumsensing AHLs. Quorum-sensing is an integral part of microbial interaction and is responsible for virulence or pathogenicity of disease-causing bacteria (7). Modulation and perturbation of this quorum-sensing pathway has been demonstrated in principle to be an effective "anti-microb...
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