Shy is a Proteobacterial steroid hydratase which catalyzes steroid side chain degradation without requiring a catalytically inert partner domain.

Shy (side chain hydratase) and Sal (side chain aldolase), are involved in successive reactions in the pathway of bile acid side chain catabolism in Proteobacteria. Untagged Shy co-purified with His-tagged Sal indicating that the two enzymes form a complex. Shy contains a MaoC and a DUF35 domain. When co-expressed with Sal, the DUF35 domain but not the MaoC domain of Shy was observed to co-purify with Sal, indicating Sal interacts with Shy through its DUF35 domain. The MaoC domain of Shy (ShyMaoC) remained catalytically viable and could hydrate cholyl-enoyl-CoA with similar catalytic efficiency as in the Shy-Sal complex. Sal expressed with the DUF35 domain of Shy (Sal-ShyDUF35) was similarly competent for the retroaldol cleavage of cholyl-3-OH-CoA. ShyMaoC showed a preference for C5 side chain bile acid substrates, exhibiting low activity towards C3 side chain substrates. The ShyMaoC structure was determined by X-ray crystallography, showing a hot dog fold with a short central helix surrounded by a twisted anti-parallel ß-sheet. Modeling and mutagenesis studies suggest that the bile acid substrate occupies the large open cleft formed by the truncated central helix and repositioning of the active site housing. ShyMaoC therefore contains two substrate binding sites per homodimer, making it distinct from previously characterized MaoC steroid hydratases that are (pseudo)-heterodimers with one substrate binding site per dimer. The characterization of Shy provides insight into how MaoC family hydratases have adapted to accommodate large polycyclic substrates that can facilitate future engineering of these enzymes to produce novel steroid pharmaceuticals.

Mechanism and linkage specificities of the dual retaining ß-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis.

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2→4) and ß-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

The retaining ß-Kdo glycosyltransferase WbbB uses a double-displacement mechanism with an intermediate adduct rearrangement step.

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 ß-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-ß-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.

Bibenzyl synthesis in Cannabis sativa L.

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.

Evidence from stable-isotope labeling that catechol is an intermediate in salicylic acid catabolism in the flowers of Silene latifolia (white campion).

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. The phytohormone salicylic acid (SA) plays a central role in the overall plant defense program, as well as various other aspects of plant growth and development. Although the biosynthetic steps toward SA are well documented, how SA is catabolized in plants remains poorly understood. Accordingly, in this study a series of stable isotope feeding experiments were performed with Silene latifolia (white campion) to explore possible routes of SA breakdown. S. latifolia flowers that were fed a solution of [2H6]-salicylic acid emitted the volatile and potent pollinator attractant, 1,2-dimethoxybenzene (veratrole), which contained the benzene ring-bound deuterium atoms. Extracts from these S. latifolia flowers revealed labeled catechol as a possible intermediate. After feeding flowers with [2H6]-catechol, the stable isotope was recovered in veratrole as well as its precursor, guaiacol. Addition of a trapping pool of guaiacol in combination with [2H6]-salicylic acid resulted in the accumulation of the label into catechol. Finally, we provide evidence for catechol O-methyltransferase enzyme activity in a population of S. latifolia that synthesizes veratrole from guaiacol. This activity was absent in non-veratrole emitting flowers. Taken together, these results imply the conversion of salicylic acid to veratrole in the following reaction sequence: salicylic acid > catechol > guaiacol > veratrole. This catabolic pathway for SA may also be embedded in other lineages of the plant kingdom, particularly those species which are known to accumulate catechol.

Designing Glycosyltransferase Expression Constructs for Improved Purification, Protein Yield, and Crystallization.

Glycosyltransferases in bacteria are built using only four known architectures, but this structural core is often supplemented by fusions with a wide variety of other domains, including those that help recruit them to the membrane. Structural and functional characterization of these proteins is often simplified by making a subconstruct that is better behaved in solution, and perhaps monofunctional. In this chapter we review bioinformatics tools and strategies that can be used for designing such constructs of glycosyltransferases.

The small RbcS-like domains of the ß-carboxysome structural protein CcmM bind RubisCO at a site distinct from that binding the RbcS subunit.

Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO2 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, CcmMS, joined by unstructured linkers. CcmMS 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 CcmMS domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmMS, however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmMS domains are able to bind RubisCO in vitro with 1.16 µm affinity. Two or four linked CcmMS domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmMS 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 CcmMS domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmMS bound equally tightly to a RubisCO variant in which the α/ß domain of RbcS was deleted, suggesting that CcmMS binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmMS may bind to an extended electronegative pocket between RbcL dimers.