PubMed · 2561647
The Authors give a review of the literature of the biochemistry of hydroxyapatite especially about structure and formation of its crystals.
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A Malzone, L Bottino, A Velotti. [Hydroxyapatite. 2. Biochemistry].. https://pubmed.ncbi.nlm.nih.gov/2561647/
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We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an R-factor of 16.8%. An enzyme from the beta-ketoadipate pathway, MLE catalyses the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one monomer consisting of 373 amino acid residues. MLE has two large domains and a C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a C-terminal meandering subdomain. The alpha beta-barrel domain is highly irregular. Its structure is (beta/alpha)7 beta, with the structural role of the last alpha-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain. The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes. The strand crossing angles also vary enormously, from +9 degrees to -69 degrees; the first and last strands, which close the barrel, cross at an angle of -69 degrees, making extensive strand-strand hydrogen bonding impossible. The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other alpha beta-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel beta-strands. The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules. The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions. Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior. The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively. This might account for the irregularity of the barrel. The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO- of muconolactone superimposed on the corresponding atoms of cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product.(ABSTRACT TRUNCATED AT 400 WORDS)
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A monoclinic crystal modification of GTP cyclohydrolase I (space group P2(1), a = 204.2 A, b = 210.4 A, c = 71.8 A, alpha = gamma = 90 degrees, beta = 95.8 degrees) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum. Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 degrees to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D5 symmetry. A procedure for hkl assignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in the ab plane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 A electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 A and a thickness of 65 A. The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail.