Rie Omi

Crystal structures of Delta1-piperideine-2-carboxylate/Delta1-pyrroline-2-carboxylate reductase belonging to a new family of NAD(P)H-dependent oxidoreductases: conformational change, substrate recognition, and stereochemistry of the reaction.

Δ1-Piperideine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato belongs to a novel sub-class in a large family of NAD(P)H-dependent oxidoreductases distinct from the conventional MDH/LDH superfamily characterized by the Rossmann fold. We have determined the structures of the following three forms of the enzyme: the unliganded form, the complex with NADPH, and the complex with NADPH and pyrrole-2-carboxylate at 1.55-, 1.8-, and 1.7-Å resolutions, respectively. The enzyme exists as a dimer, and the subunit consists of three domains; domain I, domain II (NADPH binding domain), and domain III. The core of the NADPH binding domain consists of a seven-stranded predominantly antiparallel β-sheet fold (which we named SESAS) that is characteristic of the new oxidoreductase family. The enzyme preference for NADPH over NADH is explained by the cofactor binding site architecture. A comparison of the overall structures revealed that the mobile domains I and III change their conformations to produce the catalytic form. This conformational change plays important roles in substrate recognition and the catalytic process. The active site structure of the catalytic form made it possible to identify the catalytic Asp:Ser:His triad and investigate the catalytic mechanism from a stereochemical point of view.

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.

X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the alpha/beta hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N(epsilon1) atom of Trp150 and the N(epsilon2) atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.

Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

DL-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (DL-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of DL-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6(3), with unit-cell parameters a = b = 186.2, c = 114.4 A. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V(M) value of 4.20-2.10 A3 Da(-1). A self-rotation function revealed peaks on the chi = 180 degrees section. X-ray data have been collected to 1.75 A resolution.

Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 Å resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.

Binding modes of DL-2-haloacid dehalogenase revealed by crystallography, modeling and isotope effects studies

Several pathways of biotic dechlorination can be found in enzymes, each characterized by different chlorine isotopic fractionation, which can thus serve as a signature of a particular mechanism. Unlike other dehalogenases, DL-2-haloacid dehalogenase, DL-DEX, converts both enantiomers of the substrate. Chlorine isotope effects for this enzyme are larger than in the case of other dehalogenases. Recently, the 3D structure of this enzyme became available and enabled us to model these isotope effects and seek their origin. We show that the elevated values of the chlorine kinetic isotope effects originate in part in the processes of binding and migration within the enzyme active site that precede the dehalogenation step.

Expression, purification and preliminary X-ray characterization of CTP synthetase from Thermus thermophilus HB8

A recombinant form of the CTP synthetase from Thermus thermophilus HB8 (tCTPs) was grown as colourless crystals by the hanging-drop vapour-diffusion technique using ammonium sulfate or sodium citrate as a precipitating agent. The crystals belong to space group I222, with unit-cell parameters a = 88.2, b = 118.9, c = 142.7 A, alpha = beta = gamma = 90 degrees, and are most likely to contain a monomer in the asymmetric unit with a V(M) value of 3.07 A(3) Da(-1). The crystals obtained from ammonium sulfate and sodium citrate solutions diffract X-rays to a resolution of 2.25 A using synchrotron X-ray sources and to a resolution of 2.35 A using Cu Kalpha X-rays from a rotating-anode generator.

Expression, purification and preliminary X-ray characterization of histidinol phosphate phosphatase

Histidinol phosphate phosphatase (HisPPase) catalyzes the eighth step of histidine biosynthesis, in which L-histidinol phosphate undergoes dephosphorylation to give histidinol. A recombinant form of the histidinol phosphate phosphatase from Thermus thermophilus HB8 has been expressed in Escherichia coli, purified and crystallized in two crystal forms by the hanging-drop vapour-diffusion technique. Crystal form I belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 84.8, b = 97.2, c = 74.9 A, and crystal form II belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 76.9, b = 157.6, c = 116.7 A. The crystals probably contain two monomers in the asymmetric unit, with V(M) values of 2.57 A(3) Da(-1) for form I and 2.96 A(3) Da(-1) for form II. X-ray data have been collected to 1.70 and 1.75 A resolution for crystal forms I and II, respectively.

Expression, purification and preliminary X-ray characterization of N-acetyl-γ-glutamyl-phosphate reductase from Thermus thermophilus HB8

N-Acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyses the NADPH-dependent reduction of N-acetyl-gamma-glutamyl phosphate to give the N-acetylglutamic semialdehyde. A recombinant form of AGPR from Thermus thermophilus HB8 has been crystallized by the hanging-drop vapour-diffusion technique using PEG 4000 as a precipitating agent. The crystals grew as colourless prisms, with unit-cell parameters a = b = 90.9, c = 139.5 A, alpha = beta = 90, gamma = 120 degrees. The crystals belong to the hexagonal space group P6(2)22 or P6(4)22 and are most likely to contain a monomer in the asymmetric unit, with a V(M) value of 2.19 A(3) Da(-1). The crystals diffract to a resolution of 2.2 A at beamline BL44B2 of SPring-8.