Novel Reaction Mechanism of GTP Cyclohydrolase I. High-Resolution X-Ray Crystallography of Thermus thermophilus HB8 Enzyme Complexed with a Transition State Analogue, the 8-Oxoguanine Derivative

Abstract

GTP cyclohydrolase I (GTPCH1) catalyzes the conversion of GTP to dihydroneopterin 3'-triphosphate. We found that an 8-oxoguanine derivative of GTP (8-oxo-GTP) strongly bound to GTPCH1 from Thermus thermophilus HB8 (tGTPCH1) as a competitive inhibitor. The affinity of 8-oxo-GTP was three orders of magnitude greater than that of GTP. These results suggest that 8-oxo-GTP is a transition state analogue of GTPCH1. We have solved the X-ray crystal structures of tGTPCH1 complexed with 8-oxo-GTP and 8-oxo-dGTP at 2.0 and 1.8 A resolution, respectively, as well as the free form of the enzyme at 2.2 A resolution. In the structure of tGTPCH1 complexed with 8-oxo-GTP or 8-oxo-dGTP, the oxygen atoms at O8 of the 8-oxoguanine groups, together with residues Cys108, His111 and Cys179, are coordinated to the zinc ion. The water molecule between Ndelta1 of His177 and N7 of 8-oxoguanine is conserved in both structures. These structural data are in accordance with one of the proposed transition states. Superimpositioning of the structures indicates the imidazole ring of His110 is rotated, implying concomitant proton transfer to the ribose ring O4'. Based on these structural data we propose a novel reaction mechanism for GTPCH1.

Zinc-Independent Folate Biosynthesis: Genetic, Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB

“…Results for the wild-type strain expressing GCYH-IA (}), the ⌬folE2 strain expressing GCYH-IA ({), the ⌬folE strain with delayed expression of GCYH-IB (■), the ⌬folE ⌬zur strain expressing GCYH-IB (OE), and the ⌬folE2 ⌬folE ⌬zur strain with no GCYH-IA or IB expression (F) are shown. [51] and E. coli [2] IA enzymes, respectively).…”

“…8. In E. coli (PDB ID 1FBX [46]) and T. thermophilus GCYH-IA (PDB ID 1WM9 [51]), Zn 2ϩ is coordinated in a distorted incomplete trigonal bipyramidal geometry by two thiol groups and a histidine side chain (Cys110, Cys181, His113 in E. coli residue numbers [Fig. 7]; Cys108, Cys179, His111 in T. thermophilus residue numbers [ Fig.…”

“…To elucidate the GTP binding site of GCYH-IB, we are conducting crystallographic analyses of complexes of N. gonorrhoeae GCYH-IB with GTP derivatives 8-oxo-GTP and 7-deaza-GTP. Meanwhile, we generated a docking model of 8-oxo-GTP onto N. gonorrhoeae GCYH-IB based on the superposition of the zinc-containing N. gonorrhoeae GCYH-IB crystal structure with that of the complex of T. thermophilus GCYH-IA with 8-oxo-GTP and zinc (PDB ID 1WUQ [51]), the only available structure of a ternary complex of GCYH-IA with zinc and a substrate derivative. Notably, the structures of GCYH-IA from a variety of sources (E. coli, T. thermophilus, human, and rat) are virtually identical (e.g., comparison of the crystal structures of human GCYH-IA without bound substrate [PDB ID 1FB1] and T. thermophilus GCYH-IA with 8-oxo-GTP yields an rmsd of 0.9 Å over 182 C ␣ atoms), and the active-site residues are invariant, so the comparison with T. thermophilus GCYH-IA should be relevant for all GCYH-IA members.…”

“…Notably, the structures of GCYH-IA from a variety of sources (E. coli, T. thermophilus, human, and rat) are virtually identical (e.g., comparison of the crystal structures of human GCYH-IA without bound substrate [PDB ID 1FB1] and T. thermophilus GCYH-IA with 8-oxo-GTP yields an rmsd of 0.9 Å over 182 C ␣ atoms), and the active-site residues are invariant, so the comparison with T. thermophilus GCYH-IA should be relevant for all GCYH-IA members. In the T. thermophilus structure, 8-oxo-GTP, an extremely tight-binding inhibitor, presumably mimics the intermediate that forms immediately following nucleophilic attack at C-8 of GTP in the first step of catalysis (51). The superposition was applied such that the T-fold core of the N-terminal module of one N. gonorrhoeae GCYH-IB subunit and the T-fold core of the C-terminal module of the adjacent subunit in the same ␤-barrel align maximally with the T-fold cores of two adjacent subunits of the unimodular T. thermophilus GCYH-IA, hence aligning the intersubunit interface that harbors the active site.…”

Structural Basis of Biological Nitrile Reduction

“…Both the QueF and the GCYH-I enzymes belong to the T-fold superfamily, and both are represented by unimodular (22) and bimodular (10) subfamilies where a module is built around a T-fold core (Fig. 8).…”