Substitution for Asn460 cripples ß-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.

Substrate initially binds to ß-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site. Structural and kinetic studies were done with ß-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower k(cat) values. Substrate affinity to N460D-ß-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-ß-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-ß-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-ß-galactosidase, the transition state is less stable due to an increased entropy penalty.

Relation between the activity of anaerobic microbial populations in oil sands tailings ponds and the sedimentation of tailings.

Oil sands tailings ponds contain a variety of anaerobic microbes, including methanogens, sulfate- and nitrate-reducing bacteria. Methanogenic activity in samples from a tailings pond and its input streams was higher with trimethylamine (TMA) than with acetate. Methanogens closely affiliated to Methanomethylovorans hollandica were found in the TMA enrichments. Tailings sedimentation increased with methanogenic activity, irrespective whether TMA or acetate was used to stimulate methanogenesis. Increased sedimentation of autoclaved tailings was observed with added pure cultures under methanogenic, as well as under nitrate-reducing conditions, but not under sulfate-reducing conditions. Scanning electron microscopy and energy-dispersive X-ray spectroscopy indicated the presence of microbes and of extracellular polymeric substances in tailings particle aggregates, especially under methanogenic and nitrate-reducing conditions. Hence different classes of microorganisms growing in tailings ponds contribute to increased tailings aggregation and sedimentation. Because addition of nitrate is known to lower methane production by methanogenic consortia, these observations offer the potential to combine lower methane emissions with improved microbially-induced tailings sedimentation.