Structural Determinants for Substrate Selectivity in Guanine Deaminase Enzymes of the Amidohydrolase Superfamily

Abstract

Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of Escherichia coli and Saccharomyces cerevisiae guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity.

Figure 1.. EcGuaD and Gud1 display the conserved TIM barrel fold characteristic of AHS members.

The structure of the EcGuaD protomer (A), and the Gud1 protomer (B) are shown. The metal ion (grey sphere) and the metal coordinating residues (shown in ball and stick) at the active site are given for reference. Superposition (C) of EcGuaD (purple) and Gud1 (pink) illustrates the structural similarity between the two (overall R.M.S.D. of 1.5 Å). Similarly, the superposition of the known structurally characterized guanine deaminases (D) demonstrates the high degree of structural conservation within this class of enzyme. Shown are the guanine deaminase from H. sapiens (PDB: 2UZ9, green); B. japonicum (PDB: 2OOD, orange); and C. acetobutylicum (PDB: 2I9U, blue). The metal ion (gray sphere) in the active site is shown for reference

Figure 2.. Structures of EcGuaD and Gud1 Oligomers.

Both EcGuaD (A) and Gud1 (B) appear as dimers in the crystal and as verified by size exclusion chromatography. Analysis of the dimer interface by PISA reveals that 12% of the total surface area is buried at the dimer interface for both proteins.

Figure 3.. Metal coordination within EcGuaD and Gud1 active sites.

A superposition of the EcGuaD and Gud1 active site (A) shows the conserved residues responsible for coordination of the zinc cation (shown in ball-and-stick representation; EcGuaD has purple carbon atoms and Gud1 has pink carbon atoms; nitrogen atoms are blue and oxygen atoms are red). The zinc ion is penta-coordinated by three histidine residues, an aspartate residue, and a water molecule (shown as a red sphere). There is also a conserved histidine residue His276 (297) in the correct orientation to bind but is not close enough to coordinate the metal (4 Å). The residues are numbered according to the EcGuaD sequence, with the corresponding residues in Gud1 indicated in parenthesis. (B) shows a schematic of the metal coordination. Residues and distances corresponding to Gud1 are indicated in parenthesis.

Figure 4.. Guanine deaminase substrate interactions.

Cocrystallization of Gud1 with hypoxanthine ligand-bound structure (A). Electron density for xanthine is from a POLDER map generated by omitting the ligand and is contoured at 3σ. The zinc metal ion is represented by a gray sphere and a water molecule by a red sphere. Carbon atoms are colored green, oxygen atoms are red, and nitrogen atoms are blue. A schematic showing the proposed network of binding interactions for guanine is shown in (B). Note that H-bond distances are those observed in the Gud1 liganded structure with xanthine. In both (A) and (B) residues are numbered according to the EcGuaD sequence with corresponding Gud1 residues indicated in parenthesis.

Figure 5.

Structures of compounds tested as possible substrates.

Figure 6.. Flexible loop region adjoining EcGuaD active site.

A structure of EcGuaD, colored by B-factors (low – blue, green, yellow, orange, red – high), shows that a loop region near the active site (boxed in red) has the highest B-factors. This loop, following β-strand 1, was disordered in our P1 structure of EcGuaD and was displaced by the ligand in the corresponding region in PDB entry 4AQL of human guanine deaminase bound to Valacycolovir. The zinc metal (gray sphere) in the active site is shown for reference.

Figure 7.. Proposed mechanism of AHS guanine deaminase.

Deprotonation of the activated water molecule by Asp327 generates a hydroxide ion. Nucleophilic attack at the C2 carbon atom leads to a tetrahedral intermediate. A proton transfer from the aspartate to Glu240 is facilitated by His276. Abstraction of a proton from the hydroxyl by Asp327, and concomitant release of ammonia, yields the final product.