Fernando Gil-Ortiz

Structure of Acetylglutamate Kinase, a Key Enzyme for Arginine Biosynthesis and a Prototype for the Amino Acid Kinase Enzyme Family, during Catalysis

N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis. The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices. In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate. The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase.

The crystal structure of the complex of PII and acetylglutamate kinase reveals how PII controls the storage of nitrogen as arginine.

Photosynthetic organisms can store nitrogen by synthesizing arginine, and, therefore, feedback inhibition of arginine synthesis must be relieved in these organisms when nitrogen is abundant. This relief is accomplished by the binding of the PII signal transduction protein to acetylglutamate kinase (NAGK), the controlling enzyme of arginine synthesis. Here, we describe the crystal structure of the complex between NAGK and PII of Synechococcus elongatus, at 2.75-Å resolution. We prove the physiological relevance of the observed interactions by site-directed mutagenesis and functional studies. The complex consists of two polar PII trimers sandwiching one ring-like hexameric NAGK (a trimer of dimers) with the threefold axes of these molecules aligned. The binding of PII favors a narrow ring conformation of the NAGK hexamer that is associated with arginine sites having low affinity for this inhibitor. Each PII subunit contacts one NAGK subunit only. The contacts map in the inner circumference of the NAGK ring and involve two surfaces of the PII subunit. One surface is on the PII body and interacts with the C-domain of the NAGK subunit, helping widen the arginine site found on the other side of this domain. The other surface is at the distal region of a protruding large loop (T-loop) that presents a novel compact shape. This loop is inserted in the interdomain crevice of the NAGK subunit, contacting mainly the N-domain, and playing key roles in anchoring PII on NAGK, in activating NAGK, and in complex formation regulation by MgATP, ADP, 2-oxoglutarate, and by phosphorylation of serine-49.

Arginine Biosynthesis in Thermotoga maritima: Characterization of the Arginine-Sensitive N-Acetyl-l-Glutamate Kinase

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of approximately 4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65 degrees C the I0.50 observed at 37 degrees C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75 degrees C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the Km for acetylglutamate triggered by the inhibitor, but at 37 degrees C arginine also substantially decreased the Vmax of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.

Improved cross-linked enzyme aggregates for the production of desacetyl β-lactam antibiotics intermediates

Cross-linked enzyme aggregates (CLEAs) are reported for the first time for a recombinant acetyl xylan esterase (AXE) from Bacillus pumilus. With this enzyme, CLEAs production was most effective using 3.2M (80%-saturation) ammonium sulfate, followed by cross-linking for 3h with 1% (v/v) glutaraldehyde. Particle size was a key determinant of the CLEAs activity. The usual method for generating particles, by short-time vortexing was highly inefficient in terms of enzyme activity yields. In contrast, the use of long-time vortexing increased activity recovery, and a novel approach consisting in the utilization of a commercial mechanical cell disruptor which is based on a reciprocating movement recovered all the enzyme activity in few seconds. In the CLEAs thus produced, the enzyme was much more resistant to shear, heat and extreme pH values than the soluble enzyme. The CLEAs were highly effective in transforming fully 7-amino cephalosporanic acid and cephalosporin C into their corresponding desacetyl derivatives, which are important advanced intermediates in the production of semisynthetic beta-lactam antibiotics. The operational stability of such CLEAs was remarkable, with a half life of 45 cycles. Therefore, the new methodology used here should decrease the industrial cost of the CLEAs, both in terms of biocatalyst production and reusability.

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 AlF4− Transition State Mimic

N-Acetyl-L-glutamate kinase (NAGK), the structural paradigm of the enzymes of the amino acid kinase family, catalyzes the phosphorylation of the gamma-COO(-) group of N-acetyl-L-glutamate (NAG) by ATP. We determine here the crystal structures of NAGK complexes with MgADP, NAG and the transition-state analog AlF(4)(-); with MgADP and NAG; and with ADP and SO(4)(2-). Comparison of these structures with that of the MgAMPPNP-NAG complex allows to delineate three successive steps during phosphoryl transfer: at the beginning, when the attacking and leaving O atoms and the P atom are imperfectly aligned and the distance between the attacking O atom and the P atom is 2.8A; midway, at the bipyramidal intermediate, with nearly perfect alignment and a distance of 2.3A; and, when the transfer is completed. The transfer occurs in line and is strongly associative, with Lys8 and Lys217 stabilizing the transition state and the leaving group, respectively, and with Lys61, in contrast with an earlier proposal, not being involved. Three water molecules found in all the complexes play, together with Asp162 and the Mg, crucial structural roles. Two glycine-rich loops (beta1-alphaA and beta2-alphaB) are also very important, moving in the different complexes in concert with the ligands, to which they are hydrogen-bonded, either locking them in place for reaction or stabilizing the transition state. The active site is too narrow to accommodate the substrates without compressing the reacting groups, and this compressive strain appears a crucial component of the catalytic mechanism of NAGK, and possibly of other enzymes of the amino acid kinase family such as carbamate kinase. Initial binding of the two substrates would require a different enzyme conformation with a wider active site, and the energy of substrate binding would be used to change the conformation of the active center, causing substrate strain towards the transition state.

The Crystal Structure of the Cephalosporin Deacetylating Enzyme Acetyl Xylan Esterase Bound to Paraoxon Explains the Low Sensitivity of This Serine Hydrolase to Organophosphate Inactivation.

Organophosphorus insecticides and nerve agents irreversibly inhibit serine hydrolase superfamily enzymes. One enzyme of this superfamily, the industrially important (for β-lactam antibiotic synthesis) AXE/CAH (acetyl xylan esterase/cephalosporin acetyl hydrolase) from the biotechnologically valuable organism Bacillus pumilus, exhibits low sensitivity to the organophosphate paraoxon (diethyl-p-nitrophenyl phosphate, also called paraoxon-ethyl), reflected in a high K(i) for it (~5 mM) and in a slow formation (t(½)~1 min) of the covalent adduct of the enzyme and for DEP (E-DEP, enzyme-diethyl phosphate, i.e. enzyme-paraoxon). The crystal structure of the E-DEP complex determined at 2.7 Å resolution (1 Å=0.1 nm) reveals strain in the active Ser¹⁸¹-bound organophosphate as a likely cause for the limited paraoxon sensitivity. The strain results from active-site-size limitation imposed by bulky conserved aromatic residues that may exclude as substrates esters having acyl groups larger than acetate. Interestingly, in the doughnut-like homohexamer of the enzyme, the six active sites are confined within a central chamber formed between two 60°-staggered trimers. The exclusive access to this chamber through a hole around the three-fold axis possibly limits the size of the xylan natural substrates. The enzyme provides a rigid scaffold for catalysis, as reflected in the lack of movement associated with paraoxon adduct formation, as revealed by comparing this adduct structure with that also determined in the present study at 1.9 Å resolution for the paraoxon-free enzyme.

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.

Glutamate-5-kinase from Escherichia coli: gene cloning, overexpression, purification and crystallization of the recombinant enzyme and preliminary X-ray studies

Glutamate-5-kinase (G5K) catalyzes the first step of proline (and, in mammals, ornithine) biosynthesis. It is a key regulatory point of these routes, since it is the subject of feedback allosteric inhibition by proline or ornithine. The Escherichia coli gene (proB) for G5K was cloned in pET22, overexpressed in E. coli, purified in a few steps in high yield to 95% homogeneity in the highly active proline-inhibitable form and was shown by cross-linking to be a tetramer. It was crystallized by the hanging-drop vapour-diffusion method at 294 K in the presence of ADP, MgCl(2) and L-glutamate using 1.6 M MgSO(4), 0.1 M KCl in 0.1 M MES pH 6.5 as the crystallization solution. The tetragonal bipyramid-shaped crystals diffracted to 2.5 A resolution using synchrotron radiation. The crystals belong to space group P4(1(3))2(1)2, with unit-cell parameters a = b = 101.1, c = 178.6 A, and contain two monomers in the asymmetric unit, with 58% solvent content.

Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Two Crystal Structures of Escherichia Coli N-Acetyl-L-Glutamate Kinase Demonstrate the Cycling between Open and Closed Conformations.

N-Acetyl-L-glutamate kinase (NAGK), the paradigm enzyme of the amino acid kinase family, catalyzes the second step of arginine biosynthesis. Although substrate binding and catalysis were clarified by the determination of four crystal structures of the homodimeric Escherichia coli enzyme (EcNAGK), we now determine 2 A resolution crystal structures of EcNAGK free from substrates or complexed with the product N-acetyl-L-glutamyl-5-phosphate (NAGP) and with sulfate, which reveal a novel, very open NAGK conformation to which substrates would associate and from which products would dissociate. In this conformation, the C-domain, which hosts most of the nucleotide site, rotates approximately 24 degrees -28 degrees away from the N-domain, which hosts the acetylglutamate site, whereas the empty ATP site also exhibits some changes. One sulfate is found binding in the region where the beta-phosphate of ATP normally binds, suggesting that ATP is first anchored to the beta-phosphate site, before perfect binding by induced fit, triggering the shift to the closed conformation. In contrast, the acetylglutamate site is always well formed, although its beta-hairpin lid is found here to be mobile, being closed only in the subunit of the EcNAGK-NAGP complex that binds NAGP most strongly. Lid closure appears to increase the affinity for acetylglutamate/NAGP and to stabilize the closed enzyme conformation via lid-C-domain contacts. Our finding of NAGP bound to the open conformation confirms that this product dissociates from the open enzyme form and allows reconstruction of the active center in the ternary complex with both products, delineating the final steps of the reaction, which is shown here by site-directed mutagenesis to involve centrally the invariant residue Gly11.