Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur containing cofactor, indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by Lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential action of Lipoate protein ligase B (LipB) and Lipoyl synthase (LipA). LipB uptakes octanoyl- chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. The molecular basis of substrate recognition by LipB is still not fully understood. Using E. coli LipB as a model enzyme, we show that an octanoyl-transferase mainly recognizes the 4-phosphopantetheine tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. LipB can accept octanoate- from its own ACP, noncognate ACPs, as well as C8-CoA. Further, our NMR studies demonstrate the presence of an adenine and phosphate binding site in LipB, akin to LplA. A loop containing 71RGG73 sequence, analogous to the lipoate binding loop of LplA is also conserved in LipB. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge might be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

Mimicking Enzymes: Taking Advantage of the Substrate-Recognition Properties of Metalloporphyrins in Supramolecular Catalysis

The present review describes the most relevant advances dealing with supramolecular catalysis in which metalloporphyrins are employed as substrate-recognition sites in the second coordination sphere of the catalyst. The kinetically-labile interaction between metalloporphyrins (typically, those derived from zinc) and nitrogen- or oxygen-containing substrates is energetically comparable to those non-covalent interactions (i.e. hydrogen bonding) found in enzymes enabling substrate-preorganization. Much inspired from this host-guest phenomena, the catalytic systems described in this account display unique activities, selectivities and action modes difficult to reach applying purely covalent strategies.

A shape-shifting nuclease unravels structured RNA

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3′-5′ exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3′-uridylated RNAs1-5. While the basis for Dis3L2 substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 Å. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. Typically, such modifications are incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. However, loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis (Mtb) becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. How TlyA accomplishes this feat of dual-target molecular recognition is currently unknown. Here, we report the structure of the Mtb 50S-TlyA subunit complex trapped in a post-catalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. This structure, together with complementary site-directed mutagenesis and methyltransferase functional analyses, reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144 which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. This work reveals critical aspects of substrate recognition by TlyA and suggests that base flipping is likely a common strategy among rRNA methyltransferase enzymes even in cases where the target site is accessible without such structural reorganization.

Crystal structures of glycogen-debranching enzyme mutants in complex with oligosaccharides

Debranching is a critical step in the mobilization of the important energy store glycogen. In eukaryotes, including fungi and animals, the highly conserved glycogen-debranching enzyme (GDE) debranches glycogen by a glucanotransferase (GT) reaction followed by a glucosidase (GC) reaction. Previous work indicated that these reactions are catalyzed by two active sites located more than 50 Å apart and provided insights into their catalytic mechanisms and substrate recognition. Here, five crystal structures of GDE in complex with oligosaccharides with 4–9 glucose residues are presented. The data suggest that the glycogen main chain plays a critical role in binding to the GT and GC active sites of GDE and that a minimum of five main-chain residues are required for optimal binding.