Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from<i>Sinorhizobium meliloti</i>CECT4114

Martínez‐Rodríguez, Sergio; González-Ramírez, Luis A.; Clemente-Jiménez, Josefa Marı́a; Rodríguez‐Vico, Felipe; Heras‐Vázquez, Francisco Javier Las; Gavira, José A.; García‐Ruiz, Juan Manuel

doi:10.1107/s1744309107066122

Cited by 6 publications

(2 citation statements)

References 23 publications

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“…For working at low temperatures, the mounting of the samples in capillaries became impractical due to the low rate of heat transfer. In spite of this, there are ongoing efforts to also use capillaries, in particular from counter-diffusion crystallization experiments, for low-temperature diffraction work 7,8 , but, irrespective of that, it became the standard approach in macromolecular crystallography to mount macromolecular crystals held by a thin film of mother liquor inside a thin wired loop 9,10 . Even though a number of improvements (e.g., the introduction of lithographic loops and similar structures 11 ) have been made over time to this loop-based mounting, the basic principles that were developed in the early 1990s are still in use today.…”

Section: Introductionmentioning

confidence: 99%

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Feiler

Wallacher

Weiss

2019

JoVE

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Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

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Section: Introductionmentioning

confidence: 99%

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Feiler

Wallacher

Weiss

2019

JoVE

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“…The enzyme allantoin racemase (AllR, EC 5.1.99.3) has been associated with this important function, based on both genetic and structural evidence . In particular, AllR from Klebsiella pneumoniae was crystallized, revealing close sequence and structural similarities to hydantoin racemase (HydR, EC 5.1.99.5). On the basis of biochemical evidence, both enzymes have been included in the family of cofactor-independent racemases, since both catalyze racemization of 5-substituted hydantoins without the assistance of any metal or cofactor.…”

Section: Introductionmentioning

confidence: 99%

Reaction Mechanism and Catalytic Fingerprint of Allantoin Racemase

et al. 2014

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The stereospecific oxidative decomposition of urate into allantoin is the core of purine catabolism in many organisms. The spontaneous decomposition of upstream intermediates and the nonenzymatic racemization of allantoin lead to an accumulation of (R)-allantoin, because the enzymes converting allantoin into allantoate are specific for the (S) isomer. The enzyme allantoin racemase catalyzes the reversible conversion between the two allantoin enantiomers, thus ensuring the overall efficiency of the catabolic pathway and preventing allantoin accumulation. On the basis of recent crystallographic and biochemical evidence, allantoin racemase has been assigned to the family of cofactor-independent racemases, together with other amino acid racemases. A detailed computational investigation of allantoin racemase has been carried out to complement the available experimental data and to provide atomistic insight into the enzymatic action. Allantoin, the natural substrate of the enzyme, has been investigated at the quantum mechanical level, in order to rationalize its conformational and tautomeric equilibria, playing a key role in protein-ligand recognition and in the following catalytic steps. The reaction mechanism of the enzyme has been elucidated through quantum mechanics/molecular mechanics (QM/MM) calculations. The potential energy surface investigation, carried out at the QM/MM level, revealed a stepwise reaction mechanism. A pair of cysteine residues promotes the stereoinversion of a carbon atom of the ligand without the assistance of cofactors. Electrostatic fingerprint calculations are used to discuss the role of the active site residues in lowering the pK of the substrate. The planar unprotonated intermediate is compared with the enolic allantoin tautomer observed in the active site of the crystallized enzyme. Finally, the enzymatic catalysis featured by allantoin racemase (AllR) is compared with that of other enzymes belonging to the same family.

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hydantoin racemase 5.1.99.5

Schomburg¹,

Schomburg²

2013

Class 3.4–6 Hydrolases, Lyases, Isomerases, Ligases

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Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase fromSinorhizobium melilotiCECT4114

Cited by 6 publications

References 23 publications

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Reaction Mechanism and Catalytic Fingerprint of Allantoin Racemase

hydantoin racemase 5.1.99.5

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