No abstract

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.

Enzyme activity measurement showed that L-ascorbic acid (vitamin C (Vc)) competitively inhibits the hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. The complex crystal structure of this enzyme with Vc was determined at 2.0 Å resolution. One Vc molecule was found to bind to the active site of the enzyme. The Vc carboxyl group provides the negative charges that lead the molecule into the highly positively charged cleft of the enzyme. The Vc ring system forms hydrophobic interactions with the side chain of Trp-292, which is one of the aromatic patch residues of this enzyme responsible for the selection of the cleavage sites on the substrate chain. The binding of Vc inhibits the substrate binding at hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The high concentration of Vc in human tissues probably provides a low level of natural resistance to the pneumococcal invasion. This is the first time that Vc the direct inhibition on the bacterial "spreading factor" was reported, and Vc is also the first chemical that has been shown experimentally to have an inhibitory effect on bacterial hyaluronate lyase. These studies also highlight the possible structural requirement for the design of a stronger inhibitor of bacterial hyaluronate lyase.

The Beaufort Gyre is a key feature of the Arctic Ocean, acting as a reservoir for freshwater in the region. Depending on whether the prevailing atmospheric circulation in the Arctic is anticyclonic or cyclonic, either a net accumulation or release of freshwater occurs. The sources of freshwater to the Arctic Ocean are well established and include contributions from the North American and Eurasian Rivers, the Bering Strait Pacific water inflow, sea ice meltwater, and precipitation, but their contribution to the Beaufort Gyre freshwater accumulation varies with changes in the atmospheric circulation. Here we use a Lagrangian backward tracking technique in conjunction with the 1/12‐degree resolution Nucleus for European Modelling of the Ocean model to investigate how sources of freshwater to the Beaufort Gyre have changed in recent decades, focusing on increase in the Pacific water content in the gyre between the late 1980s and early 2000s. Using empirical orthogonal functions we analyze the change in the Arctic oceanic circulation that occurred between the 1980s and 2000s. We highlight a “waiting room” advective pathway that was present in the 1980s and provide evidence that this pathway was caused by a shift in the center of Ekman transport convergence in the Arctic. We discuss the role of these changes as a contributing factor to changes in the stratification, and hence potentially the biology, of the Beaufort Gyre region.