Molecular architecture of DesV from Streptomyces venezuelae: a PLP-dependent transaminase involved in the biosynthesis of the unusual sugar desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.     

Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.     

Fluorescence energy transfer between active sites in aspartate transaminase.

The method of fluorescence energy transfer has been used to measure the distance between the active sites in a dimeric enzyme, aspartate aminotransferase. The procedure involves the prior preparation of a hybrid enzyme with the natural chromophore, pyridoxal phosphate, in one subunit as the aldimine and of the reduced aldimine in the other subunit. The two active site chromophores are used as donor and acceptor of the energy transfer and a distance of 21 Å is obtained for the separation of the active sites.     

Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme

Branched-chain amino acid aminotransferase (BCAT), which has pyridoxal 5'-phosphate as a cofactor, is a key enzyme in the biosynthetic pathway of hydrophobic amino acids (leucine, isoleucine, and valine). The enzyme reversibly catalyzes the transfer of the amino group of a hydrophobic amino acid to 2-oxoglutarate to form a 2-oxo acid and glutamate. Therefore, the active site of BCAT should have a mechanism to enable recognition of an acidic amino acid as well as a hydrophobic amino acid (double substrate recognition). The three-dimensional structures of Escherichia coli BCAT (eBCAT) in complex with the acidic substrate (glutamate) and the acidic substrate analogue (glutarate) have been determined by X-ray diffraction at 1.82 and 2.15 A resolution, respectively. The enzyme is a homo hexamer, with the polypeptide chain of the subunit folded into small and large domains, and an interdomain loop. The eBCAT in complex with the natural substrate, glutamate, was assigned as a ketimine as the most probable form based upon absorption spectra of the crystal complex and the shape of the residual electron density corresponding to the cofactor-glutamate bond structure. Upon binding of an acidic substrate, the interdomain loop approaches the substrate to shield it from the solvent region, as observed in the complex with a hydrophobic substrate. Both the acidic and the hydrophobic side chains of the substrates are bound to almost the same position in the pocket of the enzyme and are identical in structure. The inner side of the pocket is mostly hydrophobic to accommodate the hydrophobic side chain but has four sites to coordinate with the gamma-carboxylate of glutamate. The mechanism for the double substrate recognition observed in eBCAT is in contrast to those in aromatic amino acid and histidinol-phosphate aminotransferases. In an aromatic amino acid aminotransferase, the acidic side chain is located at the same position as that for the aromatic side chain because of large-scale rearrangements of the hydrogen bond network. In the histidinol-phosphate aminotransferase, the acidic and basic side chains are located at different sites and interact with different residues of the disordered loop.     

The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059

Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.     

Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties

ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.     

Crystal structures of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8: induced fit and substrate recognition

The following three-dimensional structures of three forms of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8 have been determined and represent the first x-ray analysis of the enzyme: the unliganded pyridoxal 5'-phosphate form at 1.9 A resolution and two complexes with 3-phenylpropionate and alpha-keto-gamma-methylthiobutyrate at 2.35 and 2.6 A resolution, respectively. The enzyme shows high activity toward phenylalanine, tyrosine, tryptophan, kynurenine, methionine, and glutamine. The enzyme is a homodimer, and each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to fold type I, aminotransferase subclass Ib. The subclass I aminotransferases whose structures have so far been determined exhibit a large movement of the small domain region upon binding of a substrate. Similarly, the glutamine:phenylpyruvate aminotransferase undergoes a large movement in part of the small domain to close the active site. The active-site pocket has a shape and size suitable to enclose the side chain of an aromatic amino acid or that of methionine. The inner side of the pocket is mostly hydrophobic, but also has polar sites. The kynurenine complex generated by computer modeling fits the pocket of the enzyme and its hydrophilic groups interact with the polar sites of the pocket.     

Glutamine:phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8

A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2.6.1.1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2.6.1.64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2.6.1.57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases.     

Location of heme axial ligands in the cytochrome d terminal oxidase complex of Escherichia coli determined by site-directed mutagenesis

The cytochrome d terminal oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli. This membrane-bound enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Enzyme turnover generates proton and voltage gradients across the bilayer. The oxidase is a heterodimer containing 2 mol of protoheme IX and 1 or 2 mol of heme d per mol of complex. To explain the functional properties of the enzyme, a simple model has been proposed in which it is speculated that the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of quinol and the reduction of water, respectively, are catalyzed. This paper represents an initial effort to define the axial ligands of each of the three or four hemes within the amino acid sequence of the oxidase subunits. Each of the 10 histidine residues has been altered by site-directed mutagenesis with the expectation that histidine residues are likely candidates for heme ligands. Eight of the 10 histidine residues are not essential for enzyme activity, and 2 appear to function as heme axial ligands. Histidine 186 in subunit I is required for the cytochrome b558 component of the enzyme. This residue is likely to be located near the periplasmic surface of the membrane. Histidine 19, near the amino terminus of subunit I also appears to be a heme ligand. It is concluded that two of the four or five expected heme axial ligands have been tentatively identified, although further work is required to confirm these conclusions. A minimum of two additional axial ligands must be residues other than histidine.     

Large-scale purification and some properties of the mitochondrial aspartate aminotransferase from pig heart.

A method has been developed which allows isolation of 0.3--0.5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts; the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, solutions and from solutions of ammonium sulphate. The amino acid composition, N and C-terminal amino acid sequences and subunit molecular weight have been determined; these characteristic properties are compared with those of the cytoplasmic isozyme from the same source.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.     

Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis

The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71,000 on gel-filtration. The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.     

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.     

Crystal structure of guanidinoacetate methyltransferase from rat liver: a model structure of protein arginine methyltransferase

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine methyltransferases.     

The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism.

The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.     

Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate: double substrate recognition of the enzyme

Histidinol-phosphate aminotransferase (HspAT) is a key enzyme on the histidine biosynthetic pathway. HspAT catalyzes the transfer of the amino group of L-histidinol phosphate (Hsp) to 2-oxoglutarate to form imidazole acetol phosphate (IAP) and glutamate. Thus, HspAT recognizes two kinds of substrates, Hsp and glutamate (double substrate recognition). The crystal structures of native HspAT and its complexes with Hsp and N-(5'-phosphopyridoxyl)-L-glutamate have been solved and refined to R-factors of 19.7, 19.1, and 17.8% at 2.0, 2.2, and 2.3 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. Aspartate aminotransferases (AspATs) from many species were classified into aminotransferase subgroups Ia and Ib. The primary sequence of HspAT is less than 18% identical to those of Escherichia coli AspAT of subgroup Ia and Thermus thermophilus HB8 AspAT of subgroup Ib. The X-ray analysis of HspAT showed that the overall structure is significantly similar to that of AspAT of subgroup Ib rather than subgroup Ia, and the N-terminal region moves close to the active site like that of subgroup Ib AspAT upon binding of Hsp. The folding of the main-chain atoms in the active site is conserved between HspAT and the AspATs, and more than 40% of the active-site residues is also conserved. The eHspAT recognizes both Hsp and glutamate by utilizing essentially the same active-site folding as that of AspAT, conserving the essential residues for transamination reaction, and replacing and relocating some of the active-site residues. The binding sites for the phosphate and the alpha-carboxylate groups of the substrates are roughly located at the same position and those for the imidazole and gamma-carboxylate groups at the different positions. The mechanism for the double substrate recognition observed in eHspAT is in contrast to that in aromatic amino acid aminotransferase, where the recognition site for the side chain of the acidic amino acid is formed at the same position as that for the side chain of aromatic amino acids by large-scale rearrangements of the hydrogen bond networks.     

Perforation of the tunnel wall in carbamoyl phosphate synthetase derails the passage of ammonia between sequential active sites

Carbamoyl phosphate synthetase (CPS) from Escherichia coli consists of a small subunit (approximately 42 kDa) and a large subunit (approximately 118 kDa) and catalyzes the biosynthesis of carbamoyl phosphate from MgATP, bicarbonate, and glutamine. The enzyme is able to utilize external ammonia as an alternative nitrogen source when glutamine is absent. CPS contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another. Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 A. The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation. Molecular damage to the ammonia tunnel was conducted in an attempt to induce leakage of ammonia directly to the protein exterior by the creation of a perforation in the tunnel wall. A hole in the tunnel wall was made by mutation of integral amino acid residues with alanine residues. The triple mutant alphaP360A/alphaH361A/betaR265A was unable to utilize glutamine for the synthesis of carbamoyl phosphate. However, the mutant enzyme retained full catalytic activity when external ammonia was used as the nitrogen source. The synchronization of the partial reactions occurring at the three active sites observed with the wild-type CPS was seriously disrupted with the mutant enzyme when glutamine was used as a nitrogen source. Overall, the catalytic constants of the mutant were consistent with the model where the channeling of ammonia has been disrupted due to the leakage from the ammonia tunnel to the protein exterior.