The ArsQ permease and transport of the antibiotic arsinothricin.

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.

Selective Methylation by an ArsM S-Adenosylmethionine Methyltransferase from Burkholderia gladioli GSRB05 Enhances Antibiotic Production.

Arsenic methylation contributes to the formation and diversity of environmental organoarsenicals, an important process in the arsenic biogeochemical cycle. The arsM gene encoding an arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase is widely distributed in members of every kingdom. A number of ArsM enzymes have been shown to have different patterns of methylation. When incubated with inorganic As(III), Burkholderia gladioli GSRB05 has been shown to synthesize the organoarsenical antibiotic arsinothricin (AST) but does not produce either methylarsenate (MAs(V)) or dimethylarsenate (DMAs(V)). Here, we show that cells of B. gladioli GSRB05 synthesize DMAs(V) when cultured with either MAs(III) or MAs(V). Heterologous expression of the BgarsM gene in Escherichia coli conferred resistance to MAs(III) but not As(III). The cells methylate MAs(III) and the AST precursor, reduced trivalent hydroxyarsinothricin (R-AST-OH) but do not methylate inorganic As(III). Similar results were obtained with purified BgArsM. Compared with ArsM orthologs, BgArsM has an additional 37 amino acid residues in a linker region between domains. Deletion of the additional 37 residues restored As(III) methylation activity. Cells of E. coli co-expressing the BgarsL gene encoding the noncanonical radical SAM enzyme that catalyzes the synthesis of R-AST-OH together with the BgarsM gene produce much more of the antibiotic AST compared with E. coli cells co-expressing BgarsL together with the CrarsM gene from Chlamydomonas reinhardtii, which lacks the sequence for additional 37 residues. We propose that the presence of the insertion reduces the fitness of B. gladioli because it cannot detoxify inorganic arsenic but concomitantly confers an evolutionary advantage by increasing the ability to produce AST.

A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2'-O methylation of its first nucleotide.

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

A metal ion orients mRNA to ensure accurate 2'-O ribosyl methylation of the first nucleotide of the SARS-CoV-2 genome.

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in the host cell. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

Functional and structural characterization of AntR, an Sb(III) responsive transcriptional repressor.

The ant operon of the antimony-mining bacterium Comamonas testosterone JL40 confers resistance to Sb(III). The operon is transcriptionally regulated by the product of the first gene in the operon, antR. AntR is a member of ArsR/SmtB family of metal/metalloid-responsive repressors resistance. We purified and characterized C. testosterone AntR and demonstrated that it responds to metalloids in the order Sb(III) = methylarsenite (MAs(III) >> As(III)). The protein was crystallized, and the structure was solved at 2.1 Å resolution. The homodimeric structure of AntR adopts a classical ArsR/SmtB topology architecture. The protein has five cysteine residues, of which Cys103a from one monomer and Cys113b from the other monomer, are proposed to form one Sb(III) binding site, and Cys113a and Cys103b forming a second binding site. This is the first report of the structure and binding properties of a transcriptional repressor with high selectivity for environmental antimony.

Structural basis of RNA cap modification by SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

Structural Basis of RNA Cap Modification by SARS-CoV-2 Coronavirus.

The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.