Edited by Norma M. Allewell Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO 2 escape. The key to assembling this protein complex is CcmM, a multidomain protein whose C-terminal region is required for RubisCO recruitment. This CcmM region is built as a series of copies (generally 3-5) of a small domain, CcmM S , joined by unstructured linkers. CcmM S domains have weak, but significant, sequence identity to RubisCO's small subunit, RbcS, suggesting that CcmM binds RubisCO by displacing RbcS. We report here the 1.35-Å structure of the first Thermosynechococcus elongatus CcmM S domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmM S , however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmM S domains are able to bind RubisCO in vitro with 1.16 M affinity. Two or four linked CcmM S domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmM S domains attempting to simultaneously bind a single RubisCO oligomer. Size-exclusion chromatography-coupled right-angled light scattering (SEC-RALS) and native MS experiments indicated that multiple CcmM S domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmM S bound equally tightly to a RubisCO variant in which the ␣/␤ domain of RbcS was deleted, suggesting that CcmM S binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmM S may bind to an extended electronegative pocket between RbcL dimers.Cyanobacteria are oxygenic photosynthetic bacteria that, like higher plants, fix carbon dioxide using the Calvin cycle with ribulose-bisphosphate carboxylase/oxygenase (RubisCO; 2 EC 4.1.1.39) catalyzing the key inorganic carbon fixation reaction (1). RubisCO catalyzes a chemically challenging reaction made more difficult by modern low ambient CO 2 and high O 2 concentrations, the latter acting as a competing substrate that results in an unwanted side product that requires energy to recycle (2). Cyanobacteria enhance the efficiency of this reaction by expending energy to concentrate intracellular inorganic carbon using a varied set of CO 2 and HCO 3 Ϫ pumps (3). Because CO 2 is lipophilic and readily escapes through cellular membranes, cyanobacteria accumulate only HCO 3 Ϫ in the cytosol and encapsulate RubisCO behind a secondary, (relatively) CO 2impermeable protein barrier to form a carboxysome (5-7). Carbonic anhydrase (EC 4.2.1.1), the enzyme that interconverts CO 2 and HCO 3 Ϫ , is also encapsulated so that HCO 3 Ϫ pumped into the cell only evolves into CO 2 once within the carboxysome shell (8). Carboxysomes are polyphyletic with two deeply divergent lineages, termed ␣and ␤-carboxysomes. ␣-Carboxysomes, which contain form 1A RubisCO, likely originated in chemoautotrophic ␣-prot...

WbbB, a lipopolysaccharide O-antigen synthesis enzyme from Raoultella terrigena, contains an N-terminal glycosyltransferase domain with a highly modified architecture that adds a terminal β-Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue to the O-antigen saccharide, with retention of stereochemistry. We show, using mass spectrometry, that WbbB forms a covalent adduct between the catalytic nucleophile, Asp232, and Kdo. We also determine X-ray structures for the CMP-β-Kdo donor complex, for Kdo-adducts with D232N and D232C WbbB variants, for a synthetic disaccharide acceptor complex, and for a ternary complex with both a Kdo-adduct and the acceptor. Together, these structures show that the enzyme-linked Asp232-Kdo adduct rotates to reposition the Kdo into a second sub-site, which then transfers Kdo to the acceptor. Retaining glycosyltransferases were thought to use only the front-side SNi substitution mechanism; here we show that retaining glycosyltransferases can also potentially use double-displacement mechanisms, but incorporating an additional catalytic subsite requires rearrangement of the protein’s architecture.

This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the 'bibenzyls'. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this 'bibenzyl synthase' enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing 'parts prospecting' inventory that supports the rational metabolic engineering of natural product therapeutics.

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Main conclusion A stable isotope-assisted mass spectrometry-based platform was utilized to demonstrate that the plant hormone, salicylic acid, is catabolized to catechol, a widespread secondary plant compound.