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Acta Cryst. (2014). A70, C448
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The resorcinol hydroxylase is involved in the first step of the resorcinol catabolic pathway and catalyzes hydroxylation of resorcinol to hydroxyquinol. The enzyme belongs to the two-component flavin-diffusible monooxygenase family and acts in the coexistence of two components: an oxygenase and a flavin reductase. The oxygenase component hydroxylates the substrate using molecular oxygen and reduced flavin produced by the reductase. To understand the structural basis for the catalytic mechanism, we analyzed the crystal structure of the oxygenase component (GraA) from Rhizobium sp. strain MTP-10005. The GraA subunit has 409 amino acid residues. Apo-form crystals were obtained in the tetragonal space group I4122 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and K2HPO4. Holo-form crystals were obtained in the trigonal space group P3221 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and KNO3. Both structures were determined by molecular replacement and refined at 2.3 Å and 3.2 Å resolutions, respectively. GraA is a homotetramer with three molecular two-fold axes identical to crystallographic two-fold axes in the apo-form crystal. In the holo-form crystal, four tetramers exist in the asymmetric unit and each subunit binds one FAD. The subunit consists of three domains. The N-terminal domain has an α-structure mainly of antiparallel α-helices; the central domain has a β-structure of two β-sheets stacked together; the C-terminal domain has a four-helix-bundle structure of long antiparallel α-helices involved in tetramer formation. In the holo-form, the FAD is located in the space that is encompassed by these three domains. The loop region of 13 residues, which is disordered in the apo-form, is ordered and covers FAD of another subunit. The turn portion of the loop occludes the entrance of the putative active site.

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Acta Cryst. (2014). A70, C488
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Rhizobium sp. strain MTP-10005 uses the aromatic compound γ-resorcylate as a sole source of carbon and energy for growth. Resorcinol hydroxylase, which converts resorcinol to hydroxyquinol, plays an important role in the aerobic microbial catabolism of γ-resorcylate. Resorcinol hydroxylase from Rhizobium sp. strain MTP-10005 is a two-component enzyme consisting of the reductase and the monooxygenase components. The reductase component (GraD) is an oxidoreductase containing a flavin molecule as a cofactor. GraD catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the monooxygenase component GraA to hydroxylate resorcinol to hydroxyquinol. We have determined the three-dimensional structures of recombinant GraD with a bound FAD and in complex with NAD. GraD was crystallized at 293 K by the sitting-drop vapour-diffusion method using a precipitant solution containing 13 - 14% (w/v) PEG 2000, 6 - 9% (v/v) 2-propanol, 100 mM sodium citrate pH 5.6, 100 mM DTT and 200 μM FAD. The approximate dimensions of the obtained crystals were 0.1 × 0.1 × 0.15 mm3. The crystal diffracted to 1.8Å and belongs to space group P41212 with unit-cell parameters of a = b = 77.8 Å and c = 124.2 Å. The crystal structure has been determined by the molecular replacement and refined at 1.8 Å resolution. GraD exists as a homodimer, and each monomer contains an FAD. The probable binding site for NADH is covered with the N-terminal sub-domain in chain A, whereas the site is completely exposed to bulk solvent in chain B. The NAD-complex crystals were prepared by soaking the GraD crystals in the reservoir solution supplemented with NADH. The crystal diffracted to 1.8 Å, and the crystal structure was determined at 1.8 Å resolution. The Fo-Fc maps for the crystal soaked with NADH showed the electron densities corresponding to the nicotinamide ring and the adenyl moiety in chain B.

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Acta Cryst. (2014). A70, C1055
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Aspartate racemase (AspR) catalyzes the interconversion between L- and D-aspartates without PLP. The only crystal structure of the PLP-independent amino-acid racemase is now available from a hyperthermophilic archaeon. To elucidate structural features and low-temperature adaptation of the racemase group, we determined the crystal structures of AspR from Lactobacillus sakei NBRC 15893 (LsAspR), which works in the low-to-medium temperature range, and for comparison AspR from Thermococcus litoralis DSM 5473 (TlAspR), which has the maximum activity at 95 0C. LsAspR and TlAspR weree crystallized at 20 0C by the sitting-drop vapour-diffusion method using a precipitant solution of 25% (v/v) PEG-MME 550, 5% (v/v) 2-propanol and 0.1 M sodium acetate pH 4.8 and a precipitant solution of 24% (w/v) PEG1500, 0.2 M L-proline and 0.1 M HEPES pH 7.5, respectively. The structures of LsAspR and TlAspR were determined by molecular replacement and refined at 2.6 Å resolution (R=23.8%, Rfree = 31.6%) and 2.0 Å resolution (R=18.7%, Rfree = 25.0%), respectively. Both LsAspR and TlAspR molecules are homodimers with molecular two-fold axis. The subunit of each enzyme molecule comprises the N-terminal and C-terminal domains. The molecule is formed mainly by intersubunit interactions between the N-terminal α-helices and intersubunit hydrogen-bonds between the N-terminal β-sheets in the dimer interface. The active-site cleft exists between both the domains. The spatial arrangement of the strictly conserved cystein residues in the cleft reveals the Cys residues involved in the enzymatic catalysis. A structural comparison of LsAspR and TlAspR reveals structural factors probably involved in thermostability of AspR. The molecular volume, intersubunit interaction, and the number of ion pairs suggest that the LsAspR molecule is more loose than that of TlAspR.
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