3D model of human arylamine N-acetyltransferase 2: structural basis of the slow acetylator phenotype of the R64Q variant and analysis of the active-site loop

The human arylamine N-acetyltransferase NAT2 is responsible for the biotransformation of numerous arylamine drugs and carcinogens. A common polymorphism of the NAT2 gene has been associated with susceptibility to drug toxicity and various malignancies. In this study, we used the crystal structure of the Salmonella typhimurium NAT (StNAT) to construct a high-quality model of a catalytic N-terminal region of NAT2 (residues 34-131). We show that this region has a similar structure in StNAT and the human isoforms NAT1 and NAT2. Comparison of the structures of these three molecules suggests that NATs have an active-site loop with a conserved structure, which is involved in substrate recognition. Our model is consistent with previous experimental data and provides the first plausible structural basis of the effects of a common genetic polymorphism (Arg(64)-->Gln) on NAT2 activity.     

Substrate specificity of bovine cathepsin B and its inhibition by CA074, based on crystal structure refinement of the complex

The crystal structure of the bovine spleen cathepsin B (BSCB)-CA074 complex was refined to R = 0.152 using X-ray diffraction data up to 2.18 A resolution. BSCB is characterized by an extra Cys148-Cys252 disulfide bridge, as compared with rat and human CBs. Although the crystal structures of these enzymes showed similar overall folding, a difference was observed in the occluding loop, a structural element specific only to CB. Comparison of the torsion angles indicated the different flexibilities of their loop structures. The oxirane C6 atom of CA074 was covalently bonded to the Cys29 S(gamma) atom (C3-S(gamma)=1.81 A), where the S-configuration was transformed to the R-form. Concerning the oxirane carbon atom that participates in the covalent bonding with the Cys residue, an acceptable rule has been proposed. The substrate specificities at the Sn (n = 1-3) and Sn' (n=1 and 2) subsites of CB, together with the interaction features as to CA074, have been discussed in comparison with the crystal structure of the papain-CA028 (a CA074-related inhibitor) complex.     

Structure of a closed form of human malic enzyme and implications for catalytic mechanism

Malic enzymes are widely distributed in nature and have many biological functions. The crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme in a quaternary complex with NAD+, Mn++ and oxalate has been determined at 2.2 A resolution. The structures of the quaternary complex with NAD+, Mg++, tartronate or ketomalonate have been determined at 2.6 A resolution. The structures show the enzyme in a closed form in these complexes and reveal the binding modes of the cation and the inhibitors. The divalent cation is coordinated in an octahedral fashion by six ligating oxygens, two from the substrate/inhibitor, three from Glu 255, Asp 256 and Asp 279 of the enzyme, and one from a water molecule. The structural information has significant implications for the catalytic mechanism of malic enzymes and identifies Tyr 112 and Lys 183 as possible catalytic residues. Changes in tetramer organization of the enzyme are also observed in these complexes, which might be relevant for its cooperative behavior and allosteric control.     

Crystal Structure of the Human Rad9-Hus1-Rad1 Clamp

Three evolutionarily conserved proteins, Rad9, Hus1, and Rad1, form a heterotrimeric 9-1-1 complex that plays critical roles in cellular responses to DNA damage by activating checkpoints and by recruiting DNA repair enzymes to DNA lesions. We have determined the crystal structure of the human Rad9 (residues 1-272)-Hus1-Rad1 complex at 2.5 Å resolution. The 9^1-272-1-1 complex forms a closed ring, with each subunit having a similar structure. Despite its high level of similarity to proliferating cell nucleus antigen in terms of overall structure, the 9^1-272-1-1 complex exhibits notable differences in local structures, including interdomain connecting loops, H2 and H3 helices, and loops in the vicinity of the helices of each subunit. These local structural variations provide several unique features to the 9-1-1 heterotrimeric complex—including structures of intermolecular interfaces and the inner surface around the central hole, and different electrostatic potentials at and near the interdomain connecting loops of each 9-1-1 subunit—compared to the proliferating cell nucleus antigen trimer. We propose that these structural features allow the 9-1-1 complex to bind to a damaged DNA during checkpoint control and to serve as a platform for base excision repair. We also show that the 9^1-272-1-1 complex, but not the full-length 9-1-1 complex, forms a stable complex with the 5′ recessed DNA, suggesting that the C-terminal tail of Rad9 is involved in the regulation of the 9-1-1 complex in DNA binding.     

Insight into the structure of Mesorhizobium loti arylamine N-acetyltransferase 2 (MLNAT2): a biochemical and computational study

The arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic-metabolizing enzymes (XME) that catalyze the transfer of an acetyl group from acetylCoA (Ac-CoA) to arylamine, hydrazines and their N-hydroxylated metabolites. Eukaryotes may have up to three NAT isoforms, but Mesorhizobium loti is the only prokaryote with two functional NAT isoforms (MLNAT1 and MLNAT2). The three-dimensional structure of MLNAT1 has been determined (Holton, S.J., Dairou, J., Sandy, J., Rodrigues-Lima, F., Dupret, J.M., Noble, M.E.M. and Sim, E. (2005) Structure of Mesorhizobium loti arylamine N-acetyltransferase 1. Acta Cryst, F61, 14-16). No MLNAT2 crystals have yet been produced, despite the production of sufficient quantities of pure protein. Using purified recombinant MLNAT1 and MLNAT2, we showed here that MLNAT1 was intrinsically more stable than MLNAT2. To test whether different structural features could explain these differences in intrinsic stability, we constructed a high-quality homology model for MLNAT2 based on far UV-CD data. Despite low levels of sequence identity with other prokaryotic NAT enzymes ( approximately 28% identity), this model suggests that MLNAT2 adopts the characteristic three-domain NAT fold. More importantly, molecular dynamics simulations on the structures of MLNAT1 and MLNAT2 suggested that MLNAT2 was less stable than MLNAT1 due to differences in amino-acid sequence/structure features in the alpha/beta lid domain.     

Microtube batch protein crystallization: Applications to human immunodeficiency virus type 2 (HIV-2) protease and human renin

For therapeutically relevant targets, the evaluation of enzymes in complex with their inhibitors by cocrystallization and high resolution structural analysis has become a vital component of structure-driven drug design and development. Two approaches, hanging drop vapor diffusion and a novel microtube batch method, were utilized in parallel to grow crystals of recombinant HIV -2 protease and recombinant human renin in complex with inhibitors. In the case of HIV -2 protease in complex with a reduced amide inhibitor, crystallization was achieved only by the microbatch method. In the case of human renin, the addition of precipitant was required for crystal growth. The microbatch method described here is a useful supplementary or alternative approach for screening parameters and generating crystals suitable for high resolution structural analysis. © 1994 Wiley-Liss, Inc.     

Structural insights into intermediate steps in the Sir2 deacetylation reaction

Sirtuin enzymes comprise a unique class of NAD(+)-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD(+) analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

Structural basis for the specificity of the reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125

Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the beta-anomeric hydroxyl configuration from the alpha-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35-2.20-A resolution and revealed the structural aspects of its three subsites ranging from -2 to +1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a barrier formed by a kink in the loop before helix alpha10. His-319 in this loop forms a direct hydrogen bond with the beta-hydroxyl of xylose at subsite +1, contributing to the specific recognition of anomers at the reducing end.     

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. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.     

Substrate binding in protein-tyrosine phosphatase-like inositol polyphosphatases

Protein-tyrosine phosphatase-like inositol polyphosphatases are microbial enzymes that catalyze the stepwise removal of one or more phosphates from highly phosphorylated myo-inositols via a relatively ordered pathway. To understand the substrate specificity and kinetic mechanism of these enzymes we have determined high resolution, single crystal, x-ray crystallographic structures of inactive Selenomonas ruminantium PhyA in complex with myo-inositol hexa- and pentakisphosphate. These structures provide the first glimpse of a myo-inositol polyphosphatase-ligand complex consistent with its known specificity and reveal novel features of the kinetic mechanism. To complement the structural studies, fluorescent binding assays have been developed and demonstrate that the K(d) for this enzyme is several orders of magnitude lower than the K(m). Together with rapid kinetics data, these results suggest that the protein tyrosine phosphatase-like inositol polyphosphatases have a two-step, substrate-binding mechanism that facilitates catalysis.     

Structural biology of MCM helicases

The eukaryotic MCM2-7 complex is recruited onto origins of replication during the G1 phase of the cell cycle and acts as the main helicase at the replication fork during the S phase. Over the last few years a number of structural reports on MCM proteins using both electron microscopy and protein crystallography have been published. The crystal structures of two (almost) full-length archaeal homologs provide the first atomic pictures of a MCM helicase. However one of the structures is at low resolution and the other is of an inactive MCM. Moreover, both proteins are monomeric in the crystal, whereas the activity of the complex is critically dependent on oligomerization. Lower resolution structures derived from electron microscopy studies are therefore crucial to complement the crystallographic analysis and to assemble the multimeric complex that is active in the cell. A critical analysis of all the structural results elucidates the potential conformational changes and dynamic behavior of MCM helicase to provide a first insight into the gamut of molecular configurations adopted during the processes of DNA melting and unwinding.     

The active conformation of glutamate synthase and its binding to ferredoxin

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.     

Three-dimensional structures of the Mn and Mg dTDP complexes of the family GT-2 glycosyltransferase SpsA: a comparison with related NDP-sugar glycosyltransferases.

The vast majority of glycosidic-bond synthesis in nature is performed by glycosyltransferases, which use activated glycosides as the sugar donor. Typically, the activated leaving group is a nucleoside phosphate, lipid phosphate or phosphate. The nucleotide-sugar-dependent glycosyltransferases fall into over 50 sequence-based families, with the largest and most widespread family of inverting transferases named family GT-2. Here, we present the three-dimensional crystal structure of SpsA, the first and currently the only structural representative from family GT-2, in complex with both Mn-dTDP and Mg-dTDP at a resolution of 2 A. These structures reveal how SpsA and related enzymes may display nucleotide plasticity and permit a comparison of the catalytic centre of this enzyme with those from related sequence families whose three-dimensional structures have recently been determined. Family GT-2 enzymes, together with enzymes from families 7, 13 and 43, appear to form a clan of related structures with identical catalytic apparatus and reaction mechanism.     

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.     

Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.