Ferritin is important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crystallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron.

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Chloramphenicol acetyltransferase [acetyl-CoA:chloramphenicol 03-acetyltransferase; EC 2.3.1.28] is the enzyme responsible for high-level bacterial resistance to the antibiotic chloramphenicol. It catalyzes the transfer of an acetyl group from acetyl CoA to the primary hydroxyl of chloramphenicol. The x-ray crystallographic structure of the type m variant enzyme from Escherichia cofl has been determined and refined at 1.75-resolution. The enzyme is a trimer of identical subunits with a distinctive protein fold. Structure of the trimer is stabilized by a 13-pleated sheet that extends from one subunit to the next. The active site is located at the subunit interface, and the binding sites for both chloramphenicol and CoA have been characterized. Substrate binding is unusual in that the two substrates approach the active site via clefts on opposite molecular "sides." A histidine residue previously implicated in catalysis is appropriately positioned to act as a general base catalyst in the reaction.Resistance to antibiotics in pathogenic bacteria is an increasingly common phenomenon, which has serious implications for clinical medicine. The resistance is frequently achieved by enzymatically catalyzed covalent modification of the drug. For chloramphenicol, inactivation is achieved by 0-acetylation. Because the modified drug no longer binds to a bacterial ribosome, which is its normal site of action, the drug loses its effect as an antibiotic (1). The enzyme responsible for this acetylation is chloramphenicol acetyltransferase (CAT) (acetyl-CoA:chloramphenicol acetyltransferase; EC 2.3.1.28), which catalyzes transfer of an acetyl group from acetyl CoA to the primary hydroxyl (C-3) of chloramphenicol (Cm) (2-4).The CAT gene is commonly, but not exclusively, plasmidborne in natural isolates and has been found to be a component of plasmids conferring multiple drug resistance, especially in Gram-negative bacteria and the Enterobacteriaceae, in particular (5). Amino acid sequences of several variants of CAT from both Gram-positive and Gram-negative bacteria have been determined (6-11). All known variants have similar subunit molecular weights (Mr 25,000) and are highly homologous, indicating similar tertiary structure. This conclusion had been inferred from earlier studies of hybrids formed in vivo and in vitro between naturally occurring variants (12, 13 (15), and the type III variant, which has been studied by kinetic and chemical methods (16,17) and which is currently the only variant to yield crystals suitable for x-ray diffraction studies (18).We report here the three-dimensional structures of two binary complexes of CAT, one with the substrate chloramphenicol bound and the second with bound CoA (a product of the forward reaction and substrate for the reverse reaction). Structure Determination Crystals of the binary complex of the enzyme with bound chloramphenicol were obtained by microdialysis ofprotein (5 mg/ml) in 10 mM Mes, pH 6.3, against 2% (vol/vol) 2-methyl-2,4-pentandiol/10 mM Mes, pH 6.3/1 mM chloram...

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Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4-phosphopantetheine yielding 3-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.Coenzyme A (CoA) 1 is an essential cofactor in numerous biosynthetic, degradative, and energy-yielding metabolic pathways and is involved in the control of several key reactions in intermediary metabolism (1). CoA also donates the 4Ј-phosphopantetheinyl cofactor to the acyl carrier protein of the fatty acid synthase complex (2).The synthesis of CoA occurs in five steps which, utilize pantothenate (vitamin B 5 ), cysteine, and ATP (for review, see Ref.3). In all systems studied, the rate of CoA biosynthesis appears to be regulated by feedback inhibition of the first enzyme of the pathway, pantothenate kinase (4 -7). In vitro studies of pantothenate kinase from Escherichia coli showed that (a) CoA and, to a lesser extent, its acyl thioesters are competitive inhibitors with respect to ATP and (b) the K i values are within the physiological range of intracellular CoA concentrations (6). Studies of the intermediates in CoA biosynthesis have shown that both pantothenate and 4Ј-phosphopantetheine can accumulate in the cell (8). Hence, in addition to control of CoA synthesis on the level of pantothenate kinase, further modulation of flux through the pathway could occur at phosphopantetheine adenylyltransferase (PPAT), which catalyzes the penultimate step in the pathway (Fig. 1), the reversible adenylation of 4Ј-phosphopantetheine to form 3Ј-dephospho-CoA (dPCoA) and pyrophosphate (PP i ). Regulation at this step may control the reutilization of 4Ј-phosphopantetheine arising either from the turnover of the 4Ј-phosphopantetheinyl cofactor of the acyl carrier protein (8) or the cleavage of CoA by a phosphodiesterase (9).Despite the above arguments for a role in the regulation of CoA biosynthesis, PPAT has not been the subject of a detailed study. Enzymes with PPAT activity have been purified from a number of different organisms. In mammals PPAT has been shown to be part of a complex that also includes dPCoA kinase, the effector of the fina...