Chelating Picolinaldehyde Hydrazone Amides as Protecting Groups for Carboxylic Acids: Orthogonal Reactivities of Hydrazone Amides and Esters in Hydrolysis.

We report a chelating hydrazone amide as a protecting group for carboxylic acids. Unlike most esters, 2-picolinaldehyde hydrazone amides are stable under acidic or basic hydrolytic conditions. However, hydrazone amides can be easily converted to the corresponding carboxylic acids via Ni-mediated hydrolysis. Orthogonal reactivities of the hydrazone amides and representative protecting groups were verified by control experiments and peptide synthesis, demonstrating that chelating hydrazone amides are highly useful protecting groups.

Structure of ß-1,4-mannanase from the common sea hare Aplysia kurodai at 1.05†Šresolution.

ß-1,4-Mannanase (EC 3.2.1.78) catalyzes the hydrolysis of ß-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess ß-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod ß-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Šresolution. This is the first report of the three-dimensional structure of a gastropod ß-1,4-mannanase. The structure was compared with bivalve ß-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris.

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/ß)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.

X-ray structures of transferrins and related proteins.

BACKGROUND: Transferrins are a group of iron-binding proteins including serum transferrin, lactoferrin and ovotransferrin. SCOPE OF REVIEW: The structures of transferrins are discussed. GENERAL SIGNIFICANCE: The typical transferrin molecules are folded into two homologous lobes. X-ray crystallography revealed that each lobe is further divided into two similarly sized domains, and that an iron-binding site is contained within the inter-domain cleft. The six iron coordination sites are occupied by four residues and a bidentate carbonate anion. MAJOR CONCLUSIONS: The structures of the apo- and holo-forms revealed that the transferrins undergo a large-scale conformational change upon the uptake and release of irons: domains rotate as rigid bodies around a screw axis passing through inter-domain contacts. The iron-release mechanism of transferrin N-lobe is also revealed by X-ray crystallography; two basic residues in two domains form an unusual hydrogen bond in neutral pH, and the bond should be broken and facilitate iron release at a low pH of the endosome. For ovotransferrin, the iron release kinetics of two lobes correspond well with the numbers of anion binding sites found in crystal structures. The structures of transferrins bound to other metals revealed that the flexibility of the transferrin structure allows the ability to bind to other metals. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 A resolution using a twinned crystal.

Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6(3). Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33-2.3 A. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD.

Crystallization and preliminary X-ray analysis of L-azetidine-2-carboxylate hydrolase from Pseudomonas sp. strain A2C.

L-Azetidine-2-carboxylate hydrolase from Pseudomonas sp. strain A2C catalyzes a ring-opening reaction that detoxifies L-azetidine-2-carboxylate, an analogue of L-proline. Recombinant L-azetidine-2-carboxylate hydrolase was overexpressed, purified and crystallized using polyethylene glycol and magnesium acetate as precipitants. The needle-shaped crystal belonged to space group P2(1), with unit-cell parameters a = 35.6, b = 63.6, c = 54.7 A, beta = 105.5 degrees . The crystal diffracted to a resolution of 1.38 A. The calculated V(M) value was 2.2 A(3) Da(-1), suggesting that the crystal contains one enzyme subunit in the asymmetric unit.