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Abstract: The 3′ poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript stability and inhibits translation. Nonetheless, the mechanism for poly(A) recognition by the conserved deadenylase complexes Pan2–Pan3 and Ccr4–Not is poorly understood. Here we provide a model for poly(A) RNA recognition by two DEDD-family deadenylase enzymes, Pan2 and the Ccr4–Not nuclease Caf1. Crystal structures of Saccharomyces cerevisiae Pan2 in complex with RNA show that, surprisingly, Pan2 does not form canonical base-specific contacts. Instead, it recognizes the intrinsic stacked, helical conformation of poly(A) RNA. Using a fully reconstituted biochemical system, we show that disruption of this structure—for example, by incorporation of guanosine into poly(A)—inhibits deadenylation by both Pan2 and Caf1. Together, these data establish a paradigm for specific recognition of the conformation of poly(A) RNA by proteins that regulate gene expression.

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Abstract: Serine hydroxymethyltransferase (SHMT) is the major source of 1‐carbon units required for nucleotide synthesis. Humans have cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms, which are upregulated in numerous cancers, making the enzyme an attractive drug target. Here, we show that the antifolates lometrexol and pemetrexed are inhibitors of SHMT2 and solve the first SHMT2‐antifolate structures. The antifolates display large differences in their hydrogen bond networks despite their similarity. Lometrexol was found to be the best hSHMT1/2 inhibitor from a panel antifolates. Comparison of apo hSHMT1 with antifolate bound hSHMT2 indicates a highly conserved active site architecture. This structural information offers insights as to how these compounds could be improved to produce more potent and specific inhibitors of this emerging anti‐cancer drug target.

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Abstract: Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilised for filtering of the large dataset. We obtained biophysically validated hits, some which yielded complex crystal structures. In some cases, the crystallised binding mode and top scoring mode agree, while for others the alternate ligand binding orientation was found. In this contribution we combine rigid docking, MD simulations and the LIE method to calculate free energies of binding and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favourable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.

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Abstract: Biosynthesis of 6-deoxy sugars, including L-fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hex-ose nucleotide precursors as the first committed step. Here, we determined pre- and post-catalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP-mannose C5’’ and C6’’, coupled to NADP-mediated hydride transfer from C4’’ to C6’’. We show that concerted acid-base catalysis from only two active-site groups, Tyr179 and Glu157, promotes a syn 1,4-elimination from an enol (not an enolate) intermediate. We also show that the overall multistep catalytic reaction involves least position changes of enzyme and substrate groups; and that it proceeds under conserved exploitation of the basic (minimal) catalytic machinery of short-chain dehydrogenase/reductases.

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Abstract: The opium poppy, Papaver somniferum, has been a source of medicinal alkaloids since the earliest civilizations; ca. 3400 B.C. The benzylisoquinoline alkaloid noscapine is produced commercially in P. somniferum for use as a cough suppressant and it also has potential as an anticancer compound. The first committed step in the recently elucidated noscapine biosynthetic pathway involves the conversion of scoulerine to tetrahydrocolumbamine by 9-O-methylation, catalysed by O-methyltransferase 1 (PSMT1). We demonstrate, through protein structures (obtained through rational crystal engineering at resolutions from 1.5 to 1.2Å for the engineered variants) across the reaction coordinate, how domain closure allows specific methyl transfer to generate the product. SAM-dependent methyl transfer is central to myriad natural products in plants, analysis of amino-acid sequence, now taking the three-dimensional structure of PSMT1 and low identity homologs into account, begins to shed light on the structural features that govern substrate specificity in these key, ubiquitous, plant enzymes. We propose how “gatekeeper” residues can determine acceptor regiochemistry thus allowing prediction across the wide genomic resource.