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Ornithine transcarbamylase is a highly conserved enzyme in arginine blosynthesis 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 eatalyzes 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.sub.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 blosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, [N.sup.[alpha]]-acetyl- [N.sup.[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 transearbamylases.

The global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) was characterized. To this end, hydration changes and hydrodynamic changes were measured using osmotic stress and high-precision analytical gel chromatography, respectively. Experimental results showed that specific mutations can change the structural magnitude of ATCase's conformational transition without eliminating allostery, indicating the existence of plasticity in the conformational component of allostery.

The ionic characteristics of membrane interaction by prothrombin, protein Z and other vitamin K-dependent proteins were investigated to find out the importance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein. The characteristics observed indicated that vitamin K-dependent proteins bind through ion pairing that is isolated from the aqueous environment.

The functionally linked hydration changes in Escherichia coli aspartate transcarbamylase and its catalytic subunit was examined using enzymatic assays and non linear regression. Results revealed that both osmotic stress measurements and surface area calculations of aspartate transcarbamylase (ATCase) predict water release upon ligation of the trimer. Furthermore, osmotic stress data are an accurate reflection of the hydratton behavior of ATCase.

Several crystal structures of N-acetyl-L-ornithine transcarbamylase (AOTCase) from Xanthomonas campestris (xc) are determined and [super 13]C NMR spectroscopy has shown that Lys302 is post-translationally carboxylated. Activity assays with the wild-type enzyme and many mutants have shown that the post-translational modification of lysine 302 has a key role in catalysis.

Statistical thermodynamics can be used to both determine the quantitative relations between structural and energetic aspects of protein folding and characterize folding intermediates. For example, a statistical thermodynamic model on myoglobin has been tested and agreement between theoretical predictions and differential scanning calorimetry data were found. In addition, this approach has been used in studying the folding of apo-alpha-lactalbumin and phosphoglycerate kinase in the presence of guanidine-hydrogen chloride.

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. Crystal structures of these enzymes have shown that they have a common overall fold with a trimer as their basic biological unit. The monomer structures share a common CP binding site in their N-terminal domain, but have different second substrate binding sites in their C-terminal domain. The discovery of three new transcarbamylases, L-2,3-diaminopropionate transcarbamylase (DPTCase), L-2,4-diaminobutyrate transcarbamylase (DBTCase) and ureidoglycine transcarbamylase (UGTCase), demonstrates that our knowledge and understanding of the spectrum of the transcarbamylase family is still incomplete. In this review, we summarize studies on the structures and function of transcarbamylases demonstrating how structural information helps to define biological function and how small structural differences govern enzyme specificity. Such information is important for correctly annotating transcarbamylase sequences in the genome databases and for identifying new members of the transcarbamylase family. [ABSTRACT FROM AUTHOR]

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. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups. [ABSTRACT FROM AUTHOR]

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. The NAT domain with an N-terminal His tag crystallized in space group P41212, with unit-cell parameters a = b = 51.72, c = 242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a = 63.48, b = 122.34, c = 75.88 Å, β = 107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K. [ABSTRACT FROM AUTHOR]