ADP-Ribose Pyrophosphatase Reaction in Crystalline State Conducted by Consecutive Binding of Two Manganese(II) Ions as Cofactors.

Adenosine diphosphate ribose pyrophosphatase (ADPRase), a member of the Nudix family proteins, catalyzes the metal-induced and concerted general acid-base hydrolysis of ADP ribose (ADPR) into AMP and ribose-5'-phosphate (R5P). The ADPR-hydrolysis reaction of ADPRase from Thermus thermophilus HB8 (TtADPRase) requires divalent metal cations such as Mn(2+), Zn(2+), or Mg(2+) as cofactors. Here, we report the reaction pathway observed in the catalytic center of TtADPRase, based on cryo-trapping X-ray crystallography at atomic resolutions around 1.0 Å using Mn(2+) as the reaction trigger, which was soaked into TtADPRase-ADPR binary complex crystals. Integrating 11 structures along the reaction timeline, five reaction states of TtADPRase were assigned, which were ADPRase alone (E), the ADPRase-ADPR binary complex (ES), two ADPRase-ADPR-Mn(2+) reaction intermediates (ESM, ESMM), and the postreaction state (E'). Two Mn(2+) ions were inserted consecutively into the catalytic center of the ES-state and ligated by Glu86 and Glu82, which are highly conserved among the Nudix family, in the ESM- and ESMM-states. The ADPR-hydrolysis reaction was characterized by electrostatic, proximity, and orientation effects, and by preferential binding for the transition state. A new reaction mechanism is proposed, which differs from previous ones suggested from structure analyses with nonhydrolyzable substrate analogues or point-mutated ADPRases.

Reaction mechanism and molecular basis for selenium/sulfur discrimination of selenocysteine lyase.

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

Crystal structure of a homolog of mammalian serine racemase from Schizosaccharomyces pombe.

D-serine is an endogenous coagonist for the N-methyl-D-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5'-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of L-serine to yield D-serine and vice versa. The enzyme also catalyzes the dehydration of D- and L-serine. Both reactions are enhanced by Mg.ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 A resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with L-serine and D-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique "lysino-D-alanyl" residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-D-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.

Time-programmed peptide helix inversion of a synthetic metal complex triggered by an achiral NO3- anion.

Dynamic and efficient inversion of peptide helices by an achiral NO3- anion was programmed in terms of time scale from milliseconds to hours.

Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction.

Argininosuccinate synthetase reversibly catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The structures of the enzyme from Thermus thermophilus HB8 complexed with intact ATP and substrates (citrulline and aspartate) and with AMP and product (argininosuccinate) have been determined at 2.1- and 2.0-A resolution, respectively. The enzyme does not show the ATP-induced domain rotation observed in the enzyme from Escherichia coli. In the enzyme-substrate complex, the reaction sites of ATP and the bound substrates are adjacent and are sufficiently close for the reaction to proceed without the large conformational change at the domain level. The mobility of the triphosphate group in ATP and the side chain of citrulline play an important role in the catalytic action. The protonated amino group of the bound aspartate interacts with the alpha-phosphate of ATP and the ureido group of citrulline, thus stimulating the adenylation of citrulline. The enzyme-product complex explains how the citrullyl-AMP intermediate is bound to the active site. The stereochemistry of the catalysis of the enzyme is clarified on the basis of the structures of tAsS (argininosuccinate synthetase from T. thermophilus HB8) complexes.

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.

Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase.

Acyl-CoA oxidase (ACO) catalyzes the first and rate-determining step of the peroxisomal beta-oxidation of fatty acids. The crystal structure of ACO-II, which is one of two forms of rat liver ACO (ACO-I and ACO-II), has been solved and refined to an R-factor of 20.6% at 2.2-A resolution. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. The X-ray analysis showed that the overall folding of ACO-II less C-terminal 221 residues is similar to that of medium-chain acyl-CoA dehydrogenase (MCAD). However, the N-terminal alpha- and beta-domains rotate by 13 with respect to the C-terminal alpha-domain compared with those in MCAD to give a long and large crevice that accommodates the cofactor FAD and the substrate acyl-CoA. FAD is bound to the crevice between the beta- and C-terminal domains with its adenosine diphosphate portion interacting extensively with the other subunit of the molecule. The flavin ring of FAD resides at the active site with its si-face attached to the beta-domain, and is surrounded by active-site residues in a mode similar to that found in MCAD. However, the residues have weak interactions with the flavin ring due to the loss of some of the important hydrogen bonds with the flavin ring found in MCAD. The catalytic residue Glu421 in the C-terminal alpha-domain seems to be too far away from the flavin ring to abstract the alpha-proton of the substrate acyl-CoA, suggesting that the C-terminal domain moves to close the active site upon substrate binding. The pyrimidine moiety of flavin is exposed to the solvent and can readily be attacked by molecular oxygen, while that in MCAD is protected from the solvent. The crevice for binding the fatty acyl chain is 28 A long and 6 A wide, large enough to accommodate the C23 acyl chain.

Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges.

The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution. The enzyme comprises three non-identical subunits: a four-domain alpha-subunit that harbors a di-heme cytochrome c, a seven-bladed beta-propeller beta-subunit that provides part of the active site, and a small gamma-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic gamma-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue gamma-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.