Lysine 258 in aspartate aminotransferase: Enforcer of the Circe effect for amino acid substrates and the general-base catalyst for the 1,3-prototropic shift

The ureide pathway, which mediates the oxidative degradation of uric acid to (S)-allantoin, represents the late stage of purine catabolism in most organisms. The details of uric acid metabolism remained elusive until the complete pathway involving three enzymes was recently identified and characterized. However, the molecular details of the exclusive production of one enantiomer of allantoin in this pathway are still undefined. Here we report the crystal structure of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase, which catalyzes the last reaction of the pathway, in a complex with the product, (S)-allantoin, at 2.5-Å resolution. The homodimeric helical protein represents a novel structural motif and reveals that the active site in each monomer contains no cofactors, distinguishing this enzyme mechanistically from other cofactordependent decarboxylases. On the basis of structural analysis, along with site-directed mutagenesis, a mechanism for the enzyme is proposed in which a decarboxylation reaction occurs directly, and the invariant histidine residue in the OHCU decarboxylase family plays an essential role in producing (S)-allantoin through a proton transfer from the hydroxyl group at C4 to C5 at the re-face of OHCU. These results provide molecular details that address a longstanding question of how living organisms selectively produce (S)-allantoin.The purine degradation pathway has an essential role in nitrogen metabolism in most organisms. In this catabolism pathway, inosine monophosphate, which is the final product of de novo purine biosynthesis, is degraded through sequential enzymatic steps into uric acid, which contains a high level of nitrogen (1). Most organisms, including some bacteria, plants, and animals, utilize a common pathway for uric acid metabolism and produce stereospecific (S)-allantoin as the final product (2, 3). Subsequently, (S)-allantoin, which contains an even ratio of nitrogen to carbon, is used as a nitrogen source through further enzyme-dependent degradation. This metabolism of uric acid, a process named the ureide pathway, has a pivotal role in transforming the nitrogen that is fixed in leguminous plants (2, 3) and also plays a crucial role in some bacteria under nitrogen-limited conditions (4). This pathway was once thought to be executed by a single enzyme, urate oxidase (5), but recent investigations have revealed two additional enzymes in the pathway (Scheme 1). The pathway is initiated by urate oxidase, producing the unstable 5-hydroxyisourate (HIU), 3 which has a half-life of ϳ30 min in aqueous solution, followed by hydrolysis by HIU hydrolase to produce OHCU, which also undergoes spontaneous degradation (6 -8). The third enzyme, OHCU decarboxylase, was identified through the phylogenetic analysis of a whole genome and has been shown to catalyze the decarboxylation of OHCU, producing the stereospecific (S)-allantoin (8). These observations are consistent with the previous identification of two chemically distinct labile intermediates produced by urate oxid...

Section: Discussionmentioning

confidence: 99%

Structural and Functional Basis for (S)-Allantoin Formation in the Ureide Pathway

Kim

Park

Rhee

2007

“…(E)-4-(3-hydroxy-2-methyl-5-(phosphonooxymethyl)pyridin-4-yl)-2-oxobut-3-enoic acid (10) was synthesized according to the method of Schnackerz et al (11). Removal of the cofactor from TS was performed by precipitation with ammonium sulfate at low pH according to the method of Toney and Kirsch (10) except that a 100-fold molar excess of hydroxylamine was added to the enzyme to remove PLP from the Lys residue before the addition of ammonium sulfate. The apoenzyme preparation was passed through a PD-10 (GE Healthcare) column twice to remove ammonium sulfate.…”

Section: Methodsmentioning

confidence: 99%

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Murakawa

Machida

Hayashi

2011

Threonine synthase (TS), which is a pyridoxal 5-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the ␥-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at C␤ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k cat and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, ␣-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the C␤ of the PLP-␣-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the C␤ of the analog. These results indicated that the phosphate ion released from OPHS by ␥-elimination acts as the base catalyst for the addition of water at C␤ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/ deprotonation at C␥. Threonine synthase (TS)2 catalyzes the last step of the Lthreonine biosynthesis, conversion of O-phospho-L-homoserine (OPHS) into L-threonine and inorganic phosphate (1). TS is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme and, together with the L-and D-serine dehydratases, threonine dehydratase, tryptophan synthase, and cysteine synthase, constitutes the ␤-family of PLP enzymes (2, 3). Structurally, these enzymes form fold type II PLP enzymes (4). Because TS is found only in bacteria (5), yeasts (6), and plants (4, 7), it can be a target for developing antibacterial drugs (5). For this purpose, elucidation of the mechanism of action of TS is crucial.The reaction mechanism of TS is considered to be the most complicated one catalyzed by the PLP enzymes, proceeding through all the types of intermediates formed during the catalysis of the PLP enzymes (summarized in Scheme 1 (1, 3, 8, 9)). In this mechanism, OPHS reacts with the PLP-Lys aldimine (internal aldimine 1) to form the external aldimine (2) and liberates the side chain of the Lys residue (transaldimination). The intermediate 2 is then converted to the ketimine (3) via a 1,3-prototropic shift. The electron-withdrawing imino group promotes deprotonation at C␤, and after the formation of the enamine (4), the ␥-phosphate group is eliminated, yielding ␤,␥-unsaturated ketimine (5) and a phosphate ion. In 5, deprotonation at C4Ј of the cofactor and protonation at C␥ of the substrate moiety occur to form the PLP-...

“…The enzyme was exchanged into 50 mM HEPES (pH 8.0, KOH) buffer containing 5 mM DTT using a PD-10 column, and the enzyme concentration was adjusted to 30 M. Substrates were added, and spectral changes were determined. The equilibrium constants (K eq ) for hBCATm substrate reactions were evaluated by monitoring the absorption maximum changes at 418 nm and fitting the values to the following equation by nonlinear regression analysis (21),…”

Section: Methodsmentioning

confidence: 99%

Human Mitochondrial Branched Chain Aminotransferase Isozyme

Yennawar

Islam

Conway

et al. 2006

Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center. Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm catalysis. X-ray crystallography revealed that the CXXC motif, through its network of hydrogen bonds, plays a crucial role in orienting the substrate optimally for catalysis. In all structures, there were changes in the structure of the ␤-turn preceding the CXXC motif when compared with wild type protein.The N-terminal loop between residues 15 and 32 is flexible in the oxidized and mutant enzymes, the disorder greater in the oxidized protein. Disordering of the N-terminal loop disrupts the integrity of the side chain binding pocket, particularly for the branched chain side chain, less so for the dicarboxylate substrate side chain. The kinetic studies of the mutant and oxidized enzymes support the structural analysis. The kinetic results showed that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction. The oxidized enzyme was completely inactive, whereas the mutants showed limited activity. Model building of the second half-reaction substrate ␣-ketoisocaproate in the pyridoxamine 5-phosphate-hBCATm structure suggests that disruption of the CXXC center results in altered substrate orientation and deprotonation of the amino group of pyridoxamine 5-phosphate, which inhibits catalysis. Branched chain aminotransferases (BCATs)4 (EC 2.6.1.42) catalyze transamination of the branched chain amino acids, leucine, isoleucine, and valine, to their respective ␣-keto acids, ␣-ketoisocaproate, ␣-keto-␤-methylvalerate, and ␣-ketoisovalerate. In humans, there are two isozymes, a mitochondrial (hBCATm) and a cytosolic (hBCATc) form (1-6), whereas bacteria contain only a single BCAT (6). The BCAT and transamination reactions of other pyridoxal 5Ј-phosphate (PLP)-dependent enzymes follow a ping-pong Bi-Bi reaction (7,8).Amino acid 1 ϩ ␣-keto acid 2^␣ -keto acid 1 ϩ amino acid 2 REACTION 1The reaction is accompanied by interconversion of the cofactor between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms (7-12). In the first half-reaction, the PLP form of BCAT reacts with the branched chain amino acid, and the reaction proceeds through a Michaelis complex, an external aldimine, quinonoid intermediate, ketimine, and finally the PMP form of the BCAT and the branched chain ␣-keto acid product. In the Michaelis complex, the ␣-amino group of the substrate must be deprotonated to form a new Schiff base with PLP. The migration of the proton on the ␣-amino group of the substrate to the short contact pair between the ␣-carboxylate and the phosphate of PLP reduces the electrostatic repulsion between the pair and renders the ␣-amino group of the substrate deprotonated (6). The second half-reaction is the reverse of the first half-reaction.Structurally PLP-dependent enzymes are c...