Substrate binding and catalysis in carbamate kinase ascertained by crystallographic and site-directed mutagenesis studies: movements and significance of a unique globular subdomain of this key enzyme for fermentative ATP production in bacteria.

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 A resolution and containing bound MgADP, and the other at 2.1 A resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K(m)(CP) and k(cat) triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.

[1]  M. Newcomer,et al.  Crystal Structure of Fosfomycin Resistance Kinase FomA from Streptomyces wedmorensis* , 2008, Journal of Biological Chemistry.

[2]  V. Rubio,et al.  Carbamate kinase from Enterococcus faecalis and Enterococcus faecium--cloning of the genes, studies on the enzyme expressed in Escherichia coli, and sequence similarity with N-acetyl-L-glutamate kinase. , 1998, European journal of biochemistry.

[3]  V. Rubio,et al.  Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine , 2008, Protein science : a publication of the Protein Society.

[4]  D. Linstead,et al.  The pathway of arginine catabolism in the parasitic flagellate Trichomonas vaginalis. , 1983, Molecular and biochemical parasitology.

[5]  J. Wilson,et al.  The pathway of arginine catabolism in Giardia intestinalis. , 1992, Molecular and biochemical parasitology.

[6]  M. E. Jones,et al.  CARBAMYL AND ACETYL PHOSPHOKINASE ACTIVITIES OF STREPTOCOCCUS FAECALIS AND ESCHERICHIA COLI. , 1963, The Journal of biological chemistry.

[7]  A. Mildvan.  Mechanisms of signaling and related enzymes , 1997, Proteins.

[8]  C. Marco-Marín,et al.  The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis. , 2005, Journal of molecular biology.

[9]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

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

[11]  F. Gil-Ortiz,et al.  Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis. , 2002, Structure.

[12]  D. Kern,et al.  Dynamic personalities of proteins , 2007, Nature.

[13]  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.

[14]  R. Miles.  Catabolism in mollicutes. , 1992, Journal of general microbiology.

[15]  A. Abdelal.  Arginine catabolism by microorganisms. , 1979, Annual review of microbiology.

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

[17]  R. Burne,et al.  Regulation and Physiologic Significance of the Agmatine Deiminase System of Streptococcus mutans UA159 , 2006, Journal of bacteriology.

[18]  Ignacio Fita,et al.  The course of phosphorus in the reaction of N-acetyl-L-glutamate kinase, determined from the structures of crystalline complexes, including a complex with an AlF(4)(-) transition state mimic. , 2003, Journal of molecular biology.

[19]  Eva Cusa,et al.  Genetic Analysis of a Chromosomal Region Containing Genes Required for Assimilation of Allantoin Nitrogen and Linked Glyoxylate Metabolism in Escherichia coli , 1999, Journal of bacteriology.

[20]  P. Cohen,et al.  A kinetic study of the mechanism of crystalline carbamate kinase. , 1966, The Journal of biological chemistry.

[21]  V. Stalon,et al.  Enzymes of agmatine degradation and the control of their synthesis in Streptococcus faecalis , 1982, Journal of bacteriology.

[22]  V. Rubio,et al.  The Carbamoyl-phosphate Synthetase of Pyrococcus furiosus Is Enzymologically and Structurally a Carbamate Kinase* , 1999, The Journal of Biological Chemistry.

[23]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[24]  Steven Hayward,et al.  Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50. , 2002, Journal of molecular graphics & modelling.

[25]  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.

[26]  D. Stammers,et al.  Structures of R- and T-state Escherichia coli Aspartokinase III , 2006, Journal of Biological Chemistry.

[27]  Ignacio Fita,et al.  A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase. , 2007, Journal of molecular biology.