The X-ray structure of the homotetrameric lysosomal acid hydrolase, human beta-glucuronidase (332,000 Mr), has been determined at 2.6 A resolution. The tetramer has approximate dihedral symmetry and each promoter consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179-204 form a beta-hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.
Long chain hydroxy acid oxidase (LCHAO) is a member of an FMN-dependent enzyme family that oxidizes L-2-hydroxy acids to ketoacids. LCHAO is a peroxisomal enzyme, and the identity of its physiological substrate is unclear. Mandelate is the most efficient substrate known and is commonly used in the test tube. LCHAO differs from most family members in that one of the otherwise invariant active site residues is a phenylalanine (Phe23) instead of a tyrosine. We now report the crystal structure of LCHAO. It shows the same beta8alpha8 TIM barrel structure as other structurally characterized family members, e.g., spinach glycolate oxidase (GOX) and the electron transferases yeast flavocytochrome b2 (FCB2) and Pseudomonas putida mandelate dehydrogenase (MDH). Loop 4, which is mobile in other family members, is visible in part. An acetate ion is present in the active site. The flavin interacts with the protein in the same way as in the electron transferases, and not as in GOX, an unexpected observation. An interpretation is proposed to explain this difference between GOX on one hand and FCB2 and LCHAO on the other hand, which had been proposed to arise from the differences between family members in their reactivity with oxygen. A comparison of models of the substrate bound to various published structures suggests that the very different reactivity with mandelate of LCHAO, GOX, FCB2, and MDH cannot be rationalized by a hydride transfer mechanism.
Azurin from Pseudomonas putida is a blue copper protein which functions as an electron carrier. Two crystal forms of azurin were grown, one in the presence and the other in the absence of zinc acetate; each belongs to space group P21 and contains two molecules per asymmetric unit. The zinc-free crystals have cell dimensions a = 43.25, b = 50.65, c = 54.60 A, beta = 107.79 degrees, while the crystals grown from zinc-containing solution have cell dimensions a = 40.76, b = 51.22, c = 54.96 A, beta = 103.12 degrees. The latter crystals were found to have four zinc ions incorporated into the crystal lattice. Both crystal structures were solved by the molecular-replacement method using the program MERLOT. The search model was the structure of azurin from Alcaligenes denitrificans. The crystallographic R factor for native azurin is 0.169 (Rfree = 0. 257) from 8 to 1.92 A resolution, while that for zinc azurin is 0. 181 (Rfree = 0.248) from 10 to 1.6 A resolution; for each structure the root-mean-square deviation in bond lengths from ideal values is 0.007 A. In both crystal structures the Cu atom forms three strong bonds in the equatorial plane, two with Ndelta1 from His46 and His117, and one with the thiolate S atom of Cys112. Two longer axial approaches are made by the Sgamma from Met121 and the carbonyl O atom from Gly45. This results in a distorted trigonal bipyramidal co-ordination around the Cu atom. It further confirms the presence of a weak fifth bond to the copper in P. putida azurin, as with other azurin structures described at high resolution. The Ndelta1 atom of His35 is protonated, as it is in the low-pH form of azurin from Pseudomonas aeruginosa but unlike the low-pH form of the azurins from Alcaligenes denitrificans or Alcaligenes xylosoxidans. In each crystal form the two molecules of azurin in the asymmetric unit are related by a local twofold axis and form a dimer stabilized by the interaction of a pair of hydrophobic patches surrounding the partially exposed His117 side chain. In the other known azurin crystal structures, analogous dimer formation is observed, but with different relative orientations of the molecules. The four zinc ions introduced during crystallization of zinc azurin are bound to the protein and participate in five- and sixfold ligand coordination with no affect on the copper binding site. The zinc ligands are Ndelta from His, carboxylate O atoms from Asp and Glu, Ogamma from Ser and water molecules. One of the zinc ions, located on a non-crystallographic twofold axis, links the dimers of the asymmetric unit into continuous chains parallel to the crystallographic (-101) direction and is primarily responsible for the altered unit-cell parameters. Two of the other zinc ions bind to His83, one in each molecule.
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