Introduction to Biofilms Thematic Minireview Series.

The biofilms that many bacteria and fungi produce enable them to form communities, adhere tightly to surfaces, evade host immunity, and resist antibiotics. Pathogenic microorganisms that form biofilms are very difficult to eradicate and thus are a frequent source of life-threatening, hospital-acquired infections. This series of five minireviews from the Journal of Biological Chemistry provides a broad overview of our current understanding of biofilms and the challenges that remain.

From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases.

Enzymes in the transcarbamylase family catalyze the transfer of a carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate. The two best-characterized members, aspartate transcarbamylase (ATCase) and ornithine transcarbamylase (OTCase), are present in most organisms from bacteria to humans. Recently, structures of four new transcarbamylase members, N-acetyl-L-ornithine transcarbamylase (AOTCase), N-succinyl-L-ornithine transcarbamylase (SOTCase), ygeW encoded transcarbamylase (YTCase) and putrescine transcarbamylase (PTCase) have also been determined.

The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms.

N-acetylglutamate synthase (NAGS) catalyzes the production of N-acetylglutamate (NAG) from acetyl-CoA and L-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation.

Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively.

Evolution, mechanisms, and applications of intein-mediated protein splicing.

Intein-mediated protein splicing raises questions and creates opportunities in many scientific areas. Evolutionary biologists question whether inteins are primordial enzymes or simply selfish elements, whereas biochemists seek to understand how inteins work. Synthetic chemists exploit inteins in the semisynthesis of proteins with or without nonribosomal modifications, whereas biotechnologists use modified inteins in an ever increasing variety of applications.

Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms.

N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.

Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.

Structure and function of Escherichia coli RimK, an ATP-grasp fold, L-glutamyl ligase enzyme.

We report herein the crystal structure of Escherichia coli RimK at a resolution of 2.85 Å, an enzyme that catalyzes the post-translational addition of up to 15 C-terminal glutamate residues to ribosomal protein S6. The structure belongs to the ATP-grasp superfamily and is organized as a tetramer, consistent with gel filtration analysis.

Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog.

N-Acetyl-L-glutamate synthase catalyzes the conversion of AcCoA and glutamate to CoA and N-acetyl-L-glutamate (NAG), the first step of the arginine biosynthetic pathway in lower organisms. In mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase I in the urea cycle. We have previously reported the structures of NAGS from Neisseria gonorrhoeae (ngNAGS) with various substrates bound.

Thematic minireview series on circular proteins.

Circular proteins have now been discovered in all kingdoms of life and are characterized by their exceptional stability and the diversity of their biological activities, primarily in the realm of host defense functions. This thematic minireview series provides an overview of the distribution, evolution, activities, and biological synthesis of circular proteins. It also reviews approaches that biological chemists are taking to develop synthetic methods for making circular proteins in the laboratory.

Crystal structure and biochemical properties of putrescine carbamoyltransferase from Enterococcus faecalis: Assembly, active site, and allosteric regulation.

Putrescine carbamoyltransferase (PTCase) catalyzes the conversion of carbamoylputrescine to putrescine and carbamoyl phosphate (CP), a substrate of carbamate kinase (CK). The crystal structure of PTCase has been determined and refined at 3.2 Å resolution.

A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris.

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M.

Thematic minireview series on enzyme evolution in the post-genomic era.

The emergence of genomics; ongoing computational advances; and the development of large-scale sequence, structural, and functional databases have created important new interdisciplinary linkages between molecular evolution, molecular biology, and enzymology. The five minireviews in this series survey advances and challenges in this burgeoning field from complementary perspectives. The series has three major themes.

The ygeW encoded protein from Escherichia coli is a knotted ancestral catabolic transcarbamylase.

The allantoin degradation pathway in has long been thought to involve a putative novel oxamate transcarbamylase (OXTCase) that converts oxaluric acid to oxamate and carbamyl phosphate (CP), a substrate for carbamate kinase (CK). In the genome sequence of , the only gene that could encode a novel transcarbamylase is the gene. However, the recombinant protein has no transcarbamylase activity with oxamate, allantoin, or twenty five other related compounds as potential substrates.

Reversible post-translational carboxylation modulates the enzymatic activity of N-acetyl-L-ornithine transcarbamylase.

N-Acetyl-l-ornithine transcarbamylase (AOTCase), rather than ornithine transcarbamylase (OTCase), is the essential carbamylase enzyme in the arginine biosynthesis of several plant and human pathogens. The specificity of this unique enzyme provides a potential target for controlling the spread of these pathogens. Recently, several crystal structures of AOTCase from Xanthomonas campestris (xc) have been determined.

Mechanism of allosteric inhibition of N-acetyl-L-glutamate synthase by L-arginine.

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers.

The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively.

A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase.

Transcarbamylases catalyze the transfer of the carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate such as aspartate, ornithine, or putrescine. Previously, structural determination of a transcarbamylase from Xanthomonas campestris led to the discovery of a novel N-acetylornithine transcarbamylase (AOTCase) that catalyzes the carbamylation of N-acetylornithine. Recently, a novel N-succinylornithine transcarbamylase (SOTCase) from Bacteroides fragilis was identified.

Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase.

A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6(2)22, with unit-cell parameters a = b = 134.

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

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.

Structures of N-acetylornithine transcarbamoylase from Xanthomonas campestris complexed with substrates and substrate analogs imply mechanisms for substrate binding and catalysis.

N-acetyl-L-ornithine transcarbamoylase (AOTCase) is a new member of the transcarbamoylase superfamily that is essential for arginine biosynthesis in several eubacteria. We report here crystal structures of the binary complexes of AOTCase with its substrates, carbamoyl phosphate (CP) or N-acetyl-L-ornithine (AORN), and the ternary complex with CP and N-acetyl-L-norvaline. Comparison of these structures demonstrates that the substrate-binding mechanism of this novel transcarbamoylase is different from those of aspartate and ornithine transcarbamoylases, both of which show ordered substrate binding with large domain movements.

Structure and catalytic mechanism of a novel N-succinyl-L-ornithine transcarbamylase in arginine biosynthesis of Bacteroides fragilis.

A Bacteroides fragilis gene (argF'(bf)), the disruption of which renders the bacterium auxotrophic for arginine, was expressed and its recombinant protein purified and studied. The novel protein catalyzes the carbamylation of N-succinyl-L-ornithine but not L-ornithine or N-acetyl-L-ornithine, forming N-succinyl-L-citrulline. Crystal structures of this novel transcarbamylase complexed with carbamyl phosphate and N-succinyl-L-norvaline, as well as sulfate and N-succinyl-L-norvaline have been determined and refined to 2.

Acetylornithine transcarbamylase: a novel enzyme in arginine biosynthesis.

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.

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

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.