Functional and Structural Characterization of Diverse NfsB Chloramphenicol Reductase Enzymes from Human Pathogens.

Phylogenetically diverse bacteria can carry out chloramphenicol reduction, but only a single enzyme has been described that efficiently catalyzes this reaction, the NfsB nitroreductase from Haemophilus influenzae strain KW20. Here, we tested the hypothesis that some NfsB homologs function as housekeeping enzymes with the potential to become chloramphenicol resistance enzymes. We found that expression of H. influenzae and Neisseria spp. nfsB genes, but not Pasteurella multocida nfsB, allows Escherichia coli to resist chloramphenicol by nitroreduction. Mass spectrometric analysis confirmed that purified H. influenzae and N. meningitides NfsB enzymes reduce chloramphenicol to amino-chloramphenicol, while kinetics analyses supported the hypothesis that chloramphenicol reduction is a secondary activity. We combined these findings with atomic resolution structures of multiple chloramphenicol-reducing NfsB enzymes to identify potential key substrate-binding pocket residues. Our work expands the chloramphenicol reductase family and provides mechanistic insights into how a housekeeping enzyme might confer antibiotic resistance. IMPORTANCE The question of how new enzyme activities evolve is of great biological interest and, in the context of antibiotic resistance, of great medical importance. Here, we have tested the hypothesis that new antibiotic resistance mechanisms may evolve from promiscuous housekeeping enzymes that have antibiotic modification side activities. Previous work identified a Haemophilus influenzae nitroreductase housekeeping enzyme that has the ability to give Escherichia coli resistance to the antibiotic chloramphenicol by nitroreduction. Herein, we extend this work to enzymes from other Haemophilus and Neisseria strains to discover that expression of chloramphenicol reductases is sufficient to confer chloramphenicol resistance to Es. coli, confirming that chloramphenicol reductase activity is widespread across this nitroreductase family. By solving the high-resolution crystal structures of active chloramphenicol reductases, we identified residues important for this activity. Our work supports the hypothesis that housekeeping proteins possessing multiple activities can evolve into antibiotic resistance enzymes.

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase: from the apo form to ligand complexes.

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07-2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

The crystal structure of nsp10-nsp16 heterodimer from SARS-CoV-2 in complex with S-adenosylmethionine.

SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2'-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 Å. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.

Allosteric inhibitors of Mycobacterium tuberculosis tryptophan synthase.

Global dispersion of multidrug resistant bacteria is very common and evolution of antibiotic-resistance is occurring at an alarming rate, presenting a formidable challenge for humanity. The development of new therapeuthics with novel molecular targets is urgently needed. Current drugs primarily affect protein, nucleic acid, and cell wall synthesis. Metabolic pathways, including those involved in amino acid biosynthesis, have recently sparked interest in the drug discovery community as potential reservoirs of such novel targets. Tryptophan biosynthesis, utilized by bacteria but absent in humans, represents one of the currently studied processes with a therapeutic focus. It has been shown that tryptophan synthase (TrpAB) is required for survival of Mycobacterium tuberculosis in macrophages and for evading host defense, and therefore is a promising drug target. Here we present crystal structures of TrpAB with two allosteric inhibitors of M. tuberculosis tryptophan synthase that belong to sulfolane and indole-5-sulfonamide chemical scaffolds. We compare our results with previously reported structural and biochemical studies of another, azetidine-containing M. tuberculosis tryptophan synthase inhibitor. This work shows how structurally distinct ligands can occupy the same allosteric site and make specific interactions. It also highlights the potential benefit of targeting more variable allosteric sites of important metabolic enzymes.

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase.

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-ß-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.