Publications

• Expression, purification, crystallization and preliminary X-ray crystallographic studies of a novel acetylcitrulline deacetylase from Xanthomonas campestris.

  Shi D., Yu X., Roth L., Morizono H., Hathout Y., Allewell N.M., Tuchman M.

  A novel N-acetyl-L-citrulline deacetylase that is able to catalyze the hydrolysis of N-acetyl-L-citrulline to acetate and citrulline was identified from Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the monoclinic space group C2 and diffra ... >> More

  A novel N-acetyl-L-citrulline deacetylase that is able to catalyze the hydrolysis of N-acetyl-L-citrulline to acetate and citrulline was identified from Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the monoclinic space group C2 and diffract to 1.75 A resolution, with unit-cell parameters a = 94.13, b = 95.23, c = 43.61 A, beta = 93.76 degrees. Since attempts to use homologous structural models to solve the structure via molecular replacement were unsuccessful, the selenomethionine-substituted protein was prepared using an overnight auto-induction overexpression system. Selenomethionine incorporation into the protein was verified by MALDI-TOF/TOF mass-spectroscopic analysis after trypsin digestion. The crystals of the selenomethionine-substituted protein were prepared using crystallization conditions similar to those for the native protein. Multiple anomalous dispersion (MAD) data were collected at Brookhaven National Laboratory. Structure determination is under way using the MAD phasing method. << Less

  Acta Crystallogr. F 61:676-679(2005) [PubMed] [EuropePMC]

• Acetylornithine transcarbamylase: a novel enzyme in arginine biosynthesis.

  Morizono H., Cabrera-Luque J., Shi D., Gallegos R., Yamaguchi S., Yu X., Allewell N.M., Malamy M.H., Tuchman M.

  Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed ... >> More

  Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K(m) values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N(alpha)-acetyl-N(delta)-phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases. << Less

  J. Bacteriol. 188:2974-2982(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structure of a novel N-acetyl-L-citrulline deacetylase from Xanthomonas campestris.

  Shi D., Yu X., Roth L., Tuchman M., Allewell N.M.

  The structure of a novel acetylcitrulline deacetylase from the plant pathogen Xanthomonas campestris has been solved by multiple-wavelength anomalous dispersion (MAD) using crystals grown from selenomethionine-substituted protein and refined at 1.75 A resolution. The asymmetric unit of the crystal ... >> More

  The structure of a novel acetylcitrulline deacetylase from the plant pathogen Xanthomonas campestris has been solved by multiple-wavelength anomalous dispersion (MAD) using crystals grown from selenomethionine-substituted protein and refined at 1.75 A resolution. The asymmetric unit of the crystal contains one monomer consisting of two domains, a catalytic domain and a dimerization domain. The catalytic domain is able to bind a single Co(II) ion at the active site with no change in conformation. The dimerization domain forms an interface between two monomers related by a crystallographic two-fold symmetry axis. The interface is maintained by hydrophobic interactions between helices and hydrogen bonding between two beta strands that form a continuous beta sheet across the dimer interface. Because the dimers are also related by two-fold crystallographic axes, they pack together across the crystal via the dimerization domain, suggesting that higher order oligomers may form in solution. The polypeptide fold of the monomer is similar to the fold of Pseudomonas sp. carboxypeptidase G2 and Neisseria meningitidis succinyl diaminopimelate desuccinylase. Structural comparison among these enzymes allowed modeling of substrate binding and suggests a possible catalytic mechanism, in which Glu130 functions as a bifunctional general acid-base catalyst and the metal ion polarizes the carbonyl of the acetyl group. << Less

  Biophys. Chem. 126:86-93(2007) [PubMed] [EuropePMC]