Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 Angstrom. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 Angstrom. It consists of 483 amino acids, organized similarly to the known B, lichiniformis alpha alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca2+ binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 Angstrom, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the H-2(3) half-chair/E-2 envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.
The investigations of organochlorine compounds in breast milk from women living in the Stockholm region started in 1967. The present study summarises the investigations of polychlorinated biphenyls (PCBs),- naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), polybrominated diphenyl ethers (PBDEs) and pesticides (DDT, DDE, hexachlorobenzene, dieldrin) as well as methylsulfonyl metabolites of PCBs and DDE in human milk sampled during different periods up to 1997. During the course of 20-30 yr the levels of organochlorine compounds in human milk have decreased to various extent. A decrease to the half of the original concentration was attained in the range of 4-17 yr periods. On the contrary to the organochlorine compounds, the concentrations of PBDEs have increased during the period 1972-1997, indicating a doubling of the levels by 5 yr. The levels reflect the environmental contamination and background levels in the population. The accumulation and ongoing increase in the levels of PBDEs calls for immediate measures to stop the environmental pollution and human exposure to PBDEs. (C) 2000 Elsevier Science Ltd.
A new QCD analysis of deep inelastic scattering (DIS) data is presented. All available neutrino and antineutrino cross sections are reanalyzed and included in the fit, along with charged-lepton DIS and Drell–Yan data. A massive factorization scheme is used to describe the charm component of the structure functions. Next-to-leading-order parton distribution functions are provided. In particular, the strange-sea density is determined with a higher accuracy with respect to other global fits.