The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

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. The submicromolar Km of ALDH1 for all-trans retinal, and its 600-fold enhanced affinity for retinal compared to acetaldehyde, are explained by the size and shape of the substrate entrance tunnel in ALDH1. All-trans retinal fits into the active-site pocket of ALDH1, but not into the pocket of ALDH2. Two helices and one surface loop that line the tunnel are likely to have a key role in defining substrate specificity in the wider ALDH family. The relative sizes of the tunnels also suggest why the bulky alcohol aversive drug disulfiram reacts more rapidly with ALDH1 than ALDH2. The disorder of Glu268 and the observation that NAD+ binds in two distinct modes indicate that flexibility is a key facet of the enzyme reaction mechanism.

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Bacterial flagellar protein export requires an ATPase, FliI, and presumptive inhibitor, FliH. We have explored the molecular basis for FliI/FliH interaction in the human gastric pathogen Helicobacter pylori. By using bioinformatic and biochemical analyses, we showed that residues 1-18 of FliI very likely form an amphipathic ␣-helix upon interaction with FliH, and that residues 21-91 of FliI resemble the N-terminal oligomerization domain of the F 1 -ATPase catalytic subunits. A truncated FliI-(2-91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1-18 of FliI were essential for the FliI/FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal ␣-helix of the F 1 -ATPase ␣-subunit and the globular domain of the F 1 -ATPase ␦-subunit, respectively. This similarity suggests that FliH may function as a molecular stator.

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Cereal Chem. 74(4):497-501A variety of Rapid-Visco Analyser (RVA) operating conditions have been tested with starch, flour, and wholemeal for predicting the quality of wheats for the manufacture of Japanese white-salted noodles. Using starch as the substrate, an initial temperature of 60°C has been found to be optimum, and the best heating time from this initial temperature to the peak temperature of 95°C was ≈6 min. Significant correlations were found between peak viscosity of starch pastes and noodle quality under these operating conditions. For flour and wholemeal samples, the correlations were not as high as for isolated starch. The correlations with wholemeal or flour and noodle quality could be improved by the addition of α-amylase inhibitors. Measuring RVA viscosity of flour or wholemeal in the presence of silver nitrate gave viscosities which showed highly significant correlations with noodle quality. These correlations were similar to those obtained with isolated starch. It appears that the improvement is due to inhibition of the α-amylase present in grain and flour. Correlations were also observed between flour paste viscosity and alkaline noodle quality. These could be increased either by inhibiting α-amylase with silver nitrate or by pH adjustment with sodium carbonate but the change was not significant. The improvement of the correlations by α-amylase inhibitors in this sample set was not as great as observed with Japanese white noodles.