Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus ( Sau ), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau- MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau -MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau , an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb -MenD and Sau -MenD, feedback inhibition by DHNA is a conserved mechanism in Sau -MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 μM and 47 ± 5 μM, respectively, at an ATP concentration of 20 μM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.

Millions of people are infected by the Dengue and Zika viruses each year, which can result in serious illness, permanent disability or death. There are currently no FDA-approved antivirals for treating infection by these viruses. Galidesivir is an adenosine nucleoside analog which can attenuate flavivirus replication in cell-based and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases, and subsequently incorporated into viral RNA by viral RNA-dependent RNA polymerases, leading to the termination of RNA synthesis via an unknown mechanism. Here we report the direct in vitro testing of the effects of Galidesivir triphosphate on RNA synthesis by the polymerases of Dengue-2 and Zika virus. Galidesivir triphosphate was chemically synthesized and inhibition of RNA synthesis followed using a continuous fluorescence-based assay. Galidesivir triphosphate was equipotent against the polymerase activity of Dengue-2 and Zika, with IC 50 values of 42 ± 12 μM and 47 ± 5 μM, respectively. This modest potency in vitro is consistent with results previously obtained in cell-based antiviral assays and suggests that the binding affinity for Galidesivir triphosphate is similar to the natural ATP substrate that it closely mimics. The inhibition assay we have developed will allow the rapid screening of Galidesivir and related compounds against other flavivirus polymerases, and the availability of Galidesivir triphosphate will allow detailed analysis of its mechanism of action. Highlights Galidesivir triphosphate was chemically synthesized. A continuous assay detecting double-stranded RNA formation was optimized for polymerase inhibition studies. Galidesivir triphosphate has moderate potency against DENV2 and ZIKA polymerase activity. The availability of Galidesivir triphosphate will facilitate study of its mechanism of action.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Menaquinones (vitamin K2) are a family of redox-active small molecules with critical functions across all domains of life, including energy generation in bacteria and bone health in humans. The enzymes involved in menaquinone biosynthesis also have bioengineering applications and are potential antimicrobial drug targets. New insights into the essential roles of menaquinones, and their potential to cause redox-related toxicity, have highlighted the need for this pathway to be tightly controlled. Here, we provide an overview of our current understanding of the classical menaquinone biosynthesis pathway in bacteria. We also review recent discoveries on protein-level allostery and sublocalisation of membrane-bound enzymes that have provided insight into the regulation of flux through this biosynthetic pathway.

Menangle virus is a bat-borne paramyxovirus with zoonotic potential. The single-stranded RNA genome of the virus is encapsidated in a helical nucleocapsid which is the template for both transcription and genome replication. Each of these operations is performed by the viral RNA polymerase. The phosphoprotein is the non-catalytic subunit of the polymerase, and its C-terminal region enables the polymerase to engage with the nucleocapsid. Here, we report the 1H, 15N, and 13C chemical shift assignments of the C-terminal region (amino acids 267–388) of the Menangle virus phosphoprotein. This region has a bipartite character, with a highly flexible and structurally disordered sequence preceding a structured nucleocapsid-binding domain. NMR chemical shift assignment will enable the detailed characterization of the dynamic behavior of the phosphoprotein, and its functional linkage with polymerase translocation.

Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.