Background: OleT JE oxidatively decarboxylates fatty acids to produce terminal alkenes. Results: OleT JE is an efficient peroxide-dependent lipid decarboxylase, with high affinity substrate binding and the capacity to be resolubilized from precipitate in an active form. Conclusion:OleT JE has key differences in active site structure and substrate binding/mechanistic properties from related CYP152 hydroxylases. Significance: OleT JE is an efficient and robust biocatalyst with applications in biofuel production.

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6- d eoxy- D - a ltro - h eptose and GDP-6-O M e- L - g luco - h eptose in Campylobacter jejuni . However, the substrate specificity and mechanism of action of these enzymes—C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC—are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.

All domains of life make carbohydrate polymers and by anchoring them to lipid molecules they can decorate the outside of the cell. Polysaccharides are linked to proteins by glycosylation, a process found in both bacteria and in higher organisms. Bacteria do have other distinct uses for carbohydrate polymers; in gram-negative bacteria glycolipids form the outer leaflet of the outer membrane and in many pathogens (both gram-positive and gram-negative) sugar polymers are used to build a capsule or are secreted into the environment. There are parallels, but of course differences, in the biosynthesis of glycolipids between prokaryotes and eukaryotes, which occur at the membrane. The translocation of large sugar polymers across the outer membrane is unique to gram-negative bacteria. Recent progress in the molecular understanding of both the biosynthesis at the inner membrane and the translocation across the outer membrane are reviewed here.

The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae . ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo , and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.

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