Crystal Structure of Bacillus subtilis Guanine Deaminase

Liaw, Shwu-Jen; Chang, Yu-Jui; Lai, Cheng Tsung; Chang, Hui-Chuan; Chang, Gu‐Gang

doi:10.1074/jbc.m405304200

Cited by 50 publications

(68 citation statements)

References 35 publications

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“…In the ONIOM calculations, the B3LYP functional 37,38 with the 6-31G* basis set was used for the treatment of the inner layer of the system, while AM1 was used for the rest of the system. 39 To validate the ONIOM method used in this work, the Zn coordination in the imidazole-bound bGD 11 was repro-duced by the method. (Imidazole was treated at the high level, which confirms the assignment of the protonation state of the imidazole ring.)…”

Section: Resultsmentioning

confidence: 99%

“…9,10 The crystal structure of bGD has been determined at 1.17 Å resolution. 11 The enzyme belongs to the cytidine deaminase superfamily, but, surprisingly, the two subunits form an intertwined dimeric protein by domain swapping ( Figure 1A). The high thermal stability of bGD is attributed to the domain swapping.…”

Section: Introductionmentioning

confidence: 99%

“…The high thermal stability of bGD is attributed to the domain swapping. 11 The homodimer contains two active sites, with a Zn atom coordinated with His53, Cys83, and Cys86 and a water molecule in each of the active sites ( Figure 1B). The Zn coordination chemistry is very similar to that of yeast cytosine deaminase (yCD).…”

Section: Introductionmentioning

confidence: 99%

“…Two residues, Glutamate 55 and Aspartate 114, are found to play key roles in proton shuttling in the reaction. 11 One subunit is drawn in magenta, and the other in green. Both Zn atoms are drawn in orange spheres.…”

Section: Introductionmentioning

confidence: 99%

“…The R-helices and -strands are labeled according to Liaw et al (B) The active site of bGD with the bound imidazole. 11 The amino acid residues are drawn in thin lines and the bound imidazole in thick lines. The Zn atom and the three bound water molecules (Wat1, Wat2, and Wat3) are drawn in black spheres.…”

Section: Introductionmentioning

confidence: 99%

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Catalytic Mechanism of Guanine Deaminase: An ONIOM and Molecular Dynamics Study

2007

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The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the enzyme do not affect the tautomeric state of guanine, and consequently the bound guanine is a tautomer that is the most abundant in aqueous solution. Two residues, Glutamate 55 and Aspartate 114, were found to play important roles in proton shuttling in the reaction. The proposed reaction path is initiated by proton transfer from a Zn-bound water to protonate Asp114. This process may be quite complex and rather dynamic in nature, as revealed by the molecular dynamics (MD) simulations, whereby another water may bridge the Zn-bound water and Asp114, which then is eliminated by positioning of guanine in the active site. The binding of guanine stabilizes protonated Asp114 by hydrogen bond formation. Asp114 can then transfer its proton to the N3 of the bound guanine, facilitating the nucleophilic attack on C2 of the guanine by the Zn-bound hydroxide to form a tetrahedral intermediate. This occurs with a rather low barrier. Glu55 then transfers a proton from the Zn-hydroxide to the amino group of the reaction intermediate and, at this point, the C2-N2 bond has lengthened by 0.2 Å compared to guanine, making C2-N2 bond cleavage more facile. The C2-N2 bond breaks forming ammonia, with an energy barrier of ∼8.8 kcal/mol. Ammonia leaves the active site, and xanthine is freed by the cleavage of the Zn-O2 bond, with a barrier ∼8.4 kcal/mol. Along this reaction path, the highest barrier comes from C2-N2 bond cleavage, while the barrier from the cleavage of the Zn-O2 bond is slightly smaller. The Zn-O2 bond can be broken without the assistance of water during the release of xanthine.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Catalytic Mechanism of Guanine Deaminase: An ONIOM and Molecular Dynamics Study

2007

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Structural characterization of the zinc binding domain in cytosolic PSD‐95 interactor (cypin): Role of zinc binding in guanine deamination and dendrite branching

Fernández

Welsh²,

Firestein

2008

Proteins

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Dendrite morphology regulates how a postsynaptic neuron receives information from presynaptic neurons. The specific patterning of dendrite branches is promoted by extrinsic and intrinsic factors that trigger the activation of functional signaling pathways. However, most of the regulating factors and the biochemical mechanisms involved in regulating dendrite branching are unknown. Our laboratory previously reported that cypin (cytosolic PSD-95 interactor) plays an active role in regulating dendrite branching in hippocampal neurons. Cypin-promoted increases in dendrite number are dependent on guanine deaminase activity. In order to identify the specific structural role of zincbinding in cypin-mediated dendrite branching and guanine deaminase activity, we employed computational homology modeling techniques to construct a three dimensional structural model of cypin. Analysis of the protein-ion sequestration scaffold of this model identified several histidines and aspartic acid residues responsible for zinc binding. Single substitution mutations in these specific sites completely disrupted the guanine deaminase enzymatic activity and rendered cypin unable to promote dendrite branching in rat hippocampal neurons. The specific zinc ion-binding function of each residue in the protein scaffold was also confirmed by Inductively Coupled Plasma-Optic Emission Spectrometry. Inspection of our structural model confirmed that His82 and His84 coordinate with the zinc ion, together with His240, His279, and Asp330, residues that until now were unknown to play a role in this regard. Furthermore, promotion of dendrite branching by cypin is zincdependent.

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Phylogenetic Analysis and Molecular Evolution of Guanine Deaminases: From Guanine to Dendrites

Fernández

Byrne²,

Firestein

2009

J Mol Evol

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Guanine deaminase (GDA; guanase) is a ubiquitous enzyme that catalyzes the first step of purine metabolism by hydrolytic deamination of guanine, resulting in the production of xanthine. This hydrolase subfamily member plays an essential role in maintaining homeostasis of cellular triphosphate nucleotides for energy, signal transduction pathways, and nitrogen sources. In mammals, GDA protein levels can play a role in neuronal development by regulating dendritic arborization. We previously demonstrated that the most abundant alternative splice form of GDA in mammals, termed cypin (cytosolic PSD-95 interactor), interacts with postsynaptic density proteins, regulates microtubule polymerization, and increases dendrite number. Since purine metabolism and dendrite development were previously thought to be independent cellular processes, this multifunctional protein serves as a new target for the treatment of cognitive disorders characterized by aberrant neuronal morphology and purine metabolism. Although the enzymatic activity of GDA has been conserved during evolution from prokaryotes to higher eukaryotes, a detailed evolutionary assessment of the principal domains in GDA proteins has not yet been put forward. In this study, we perform a complete evolutionary analysis of the full-length sequences and the principal domains in guanine deaminases. Furthermore, we reconstruct the molecular phylogeny of guanine deaminases with neighbor-joining, maximum-likelihood, and UPGMA methods of phylogenetic inference. This study can act as a model whereby a universal housekeeping enzyme may be adapted to act also as a key regulator of a developmental process.

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Crystal Structure of Bacillus subtilis Guanine Deaminase

Cited by 50 publications

References 35 publications

Catalytic Mechanism of Guanine Deaminase: An ONIOM and Molecular Dynamics Study

Catalytic Mechanism of Guanine Deaminase: An ONIOM and Molecular Dynamics Study

Structural characterization of the zinc binding domain in cytosolic PSD‐95 interactor (cypin): Role of zinc binding in guanine deamination and dendrite branching

Phylogenetic Analysis and Molecular Evolution of Guanine Deaminases: From Guanine to Dendrites

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