Studies on the active-site of Escherichia coli shikimate dehydrogenase

Shikimate dehydrogenase (SKDH) catalyses the fourth step of the biosynthetic shikimate pathway, the reversible reduction of 3-dehydroshikimate to shikimate. This Thesis describes the work leading to the identification of three active-site residues in E. coli SKDH. Group specific chemical modification with trinitrobenzene sulfonic acid (TNBS) and kinetic analyses indicated the presence of an essential lysine residue at the active-site. Electrospray mass spectrometry (ESMS) revealed that three lysine residues were modified by TNBS, and in the presence of substrate and coenzyme two were protected from modification. HPLC mass spectrometry (LCMS) identified the three modified residues as Lys-15, Lys- 65 and Lys-217/219. In the presence of substrate and coenzyme Lys-65 was completely protected and Lys-15 was partially protected from modification. Sequence comparison with other known SKDH sequences allowed the identification of Lys-65 as the essential lysine residue. Experiments with methyl shikimate have provided evidence for a role for Lys-65 in substrate binding. Arg-154 was identified as a component of the coenzyme (NADP+) binding site by group specific chemical modification with phenylglyoxal (PGO). Characterisation of PGO modified SKDH by ESMS showed that a PGO modified arginine residue could have either a 1:1 or 2:1 stoichiometry. Protection experiments suggest that Arg-154 interacts with the 2' phosphate group of the adenosine moiety of the coenzyme. Chemical modification with the group specific reagent diethylpyrocarbonate (DEPC) and kinetic analyses indicated the presence of an active-site histidine. Characterisation of DEPC modified SKDH by ESMS showed that in the presence of substrate and coenzyme two histidine residues were protected from DEPC modification. Differential peptide mapping using reverse phase HPLC identified the two protected residues as His-13 and His-253. Sequence comparison with other SKDH sequences identified His-13 as the essential histidine. PH-dependence studies indicated a role for His-13 as a general acid/base in the catalytic reaction of SKDH. As a preliminary step towards solving the three dimensional structure of SKDH small crystals have been obtained

Crystallization and preliminary X-ray analysis of shikimate dehydrogenase fromEscherichia coli

Shikimate dehydrogenase from Escherichia coli has been crystallized by the vapour-diffusion method using ammonium sulfate as a precipitant. Mass spectrometry confirmed the purity of the enzyme and dynamic light scattering was used to find the appropriate additives to yield a monodisperse enzyme solution. The crystals are monoclinic, space group C2, with unit-cell parameters a = 110.0, b = 139.8, c = 102.6 Å, [beta] = 122.2° (at 100 K). Native crystals diffract to 2.3 Å in-house on a rotating-anode X-ray source. The asymmetric unit is likely to contain four molecules, related by 222 symmetry, corresponding to a packing density of 2.86 Å3 Da-1

Physical activity tracking among Sri Lankan adults: findings from a 7-year follow-up of the Ragama Health Study

Limited data are available on physical activity tracking among adults in low- and middle-income countries. Using a longitudinal design, we assessed trends and correlates of physical activity among Sri Lankan adults. Individuals selected through age-stratified random sampling, were screened initially in 2007 (n = 2986) and reevaluated in 2014 (n = 2148). On both occasions, structured interviews and clinical measurements were completed. Approximately 40% of the participants engaged in recommended levels of physical activity both at baseline and follow-up. One-fifth reported increased physical activity at follow-up, a similar proportion reported being persistently inactive or a reduction in physical activity. In the adjusted analysis, being persistently active was associated with male sex, a lower educational level and income, being free of any chronic disease conditions, better self-rated health, and sitting time <8 hours. Our findings support public health interventions to help maintain recommended physical activity levels over time, particularly for subgroups at high-risk of physical inactivity