Crystal structure of a transcarbamylase-like protein from the anaerobic bacterium Bacteroides fragilis at 2.0 A resolution.

A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4(3)2(1)2 (a=b=153.4 A, c=94.8 A). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 A resolution to an R-factor of 20.6% (R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel beta-sheet surrounded by alpha-helices and loops with alpha/beta topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.

[1]  M. Turner,et al.  An adenosine triphosphate-dependent carbamoylphosphate--3-hydroxymethylcephem O-carbamoyltransferase from Streptomyces clavuligerus. , 1980, The Biochemical journal.

[2]  R. Wolfe,et al.  OXAMIC TRANSCARBAMYLASE OF STREPTOCOCCUS ALLANTOICUS , 1964, Journal of bacteriology.

[3]  T. Alber,et al.  Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Ōmura,et al.  A new antifungal antibiotic, 3'-O-decarbamoylirumamycin. , 1985, The Journal of antibiotics.

[5]  D. A. Palmieri,et al.  The genome sequence of the plant pathogen Xylella fastidiosa , 2000, Nature.

[6]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[7]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[8]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[9]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[10]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[11]  E. Kantrowitz,et al.  An improved colorimetric assay for aspartate and ornithine transcarbamylases. , 1981, Analytical biochemistry.

[12]  N. Glansdorff,et al.  The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Shi,et al.  Crystal structure of human ornithine transcarbamylase complexed with carbamoyl phosphate and L‐norvaline at 1.9 Å resolution , 2000, Proteins.

[14]  N. Glansdorff,et al.  The Evolutionary History of Carbamoyltransferases: A Complex Set of Paralogous Genes Was Already Present in the Last Universal Common Ancestor , 1999, Journal of Molecular Evolution.

[15]  A. Eller,et al.  Further evidence for a separate enzymic entity for the synthesis of homocitrulline, distinct from the regular ornithine transcarbamylase. , 1984, Enzyme.

[16]  Structure and properties of the putrescine carbamoyltransferase of Streptococcus faecalis. , 1979, European journal of biochemistry.

[17]  N. Allewell,et al.  Substrate-induced conformational change in a trimeric ornithine transcarbamoylase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[19]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[20]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[21]  Mike Carson,et al.  RIBBONS 2.0 , 1991 .

[22]  J. Martín,et al.  Characterization of the cmcH genes of Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis. , 1995, Gene.

[23]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[24]  V. Stalon,et al.  Control of enzyme synthesis in the oxalurate catabolic pathway of Streptococcus faecalis ATCC 11700: evidence for the existence of a third carbamate kinase , 1986, Archives of Microbiology.

[25]  B. Seaton,et al.  Crystal structure at 2.8 Å resolution of anabolic ornithine transcarbamylase from Escherichia coli , 1997, Nature Structural Biology.

[26]  Boguslaw Stec,et al.  A cis-proline to alanine mutant of E. coli aspartate transcarbamoylase: kinetic studies and three-dimensional crystal structures. , 2000, Biochemistry.

[27]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[28]  J. Wild,et al.  Molecular evolution and genetic engineering of protein domains involving aspartate transcarbamoylase. , 1990, Annual review of microbiology.

[29]  C. Tricot,et al.  Evolutionary relationships among bacterial carbamoyltransferases. , 1989, Journal of general microbiology.

[30]  D. Shi,et al.  1.85-A resolution crystal structure of human ornithine transcarbamoylase complexed with N-phosphonacetyl-L-ornithine. Catalytic mechanism and correlation with inherited deficiency. , 1998, The Journal of biological chemistry.

[31]  T. Alber,et al.  Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: implications for allosteric regulation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  W. Lipscomb,et al.  Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N‐phosphonacetyl‐L‐aspartate at 2.1 Å , 1999, Proteins.

[33]  J. Wild,et al.  Protein differentiation: a comparison of aspartate transcarbamoylase and ornithine transcarbamoylase from Escherichia coli K-12. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[34]  B. A. Fields,et al.  Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism. , 2000, The Journal of biological chemistry.

[35]  T. Alber,et al.  Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: implications for allosteric regulation induced cell migration. , 2000 .

[36]  Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces 'late onset' hyperammonaemia. , 1997, The Biochemical journal.

[37]  Scott Df,et al.  Separation of ornithine and lysine activities of the ornithine-transcarbamylase-catalyzed reaction. , 1983 .

[38]  W. Lipscomb.  Aspartate transcarbamylase from Escherichia coli: activity and regulation. , 1994, Advances in enzymology and related areas of molecular biology.