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. 2007 May 1;46(17):5050-62.
doi: 10.1021/bi061637j. Epub 2007 Apr 4.

A novel function for the N-terminal nucleophile hydrolase fold demonstrated by the structure of an archaeal inosine monophosphate cyclohydrolase

You-Na Kang  1 , Anh Tran, Robert H White, Steven E Ealick
Affiliations

A novel function for the N-terminal nucleophile hydrolase fold demonstrated by the structure of an archaeal inosine monophosphate cyclohydrolase

You-Na Kang et al. Biochemistry. .

Abstract

Inosine 5'-monophosphate (IMP) cyclohydrolase catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) to IMP in the final step of de novo purine biosynthesis. Two major types of this enzyme have been discovered to date: PurH in Bacteria and Eukarya and PurO in Archaea. The structure of the MTH1020 gene product from Methanothermobacter thermoautotrophicus was previously solved without functional annotation but shows high amino acid sequence similarity to other PurOs. We determined the crystal structure of the MTH1020 gene product in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) at 2.0 and 2.6 A resolution, respectively. On the basis of the sequence analysis, ligand-bound structures, and biochemical data, MTH1020 is confirmed as an archaeal IMP cyclohydrolase, thus designated as MthPurO. MthPurO has a four-layered alphabeta betaalpha core structure, showing an N-terminal nucleophile (NTN) hydrolase fold. The active site is located at the deep pocket between two central beta-sheets and contains residues strictly conserved within PurOs. Comparisons of the two types of IMP cyclohydrolase, PurO and PurH, revealed that there are no similarities in sequence, structure, or the active site architecture, suggesting that they are evolutionarily not related to each other. The MjR31K mutant of PurO from Methanocaldococcus jannaschii showed 76% decreased activity and the MjE102Q mutation completely abolished enzymatic activity, suggesting that these highly conserved residues play critical roles in catalysis. Interestingly, green fluorescent protein (GFP), which has no structural homology to either PurO or PurH but catalyzes a similar intramolecular cyclohydrolase reaction required for chromophore maturation, utilizes Arg96 and Glu222 in a mechanism analogous to that of PurO.

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Figures

Figure 1
Figure 1
Two different pathways for the last two steps of de novo purine biosynthesis. In Bacteria and Eukarya, one bifunctional enzyme PurH performs these reactions, whereas some Archaea employ two separate enzymes for the same chemical reactions. PurP catalyzes the formylation of AICAR to FAICAR by using ATP and formate, and then PurO cyclizes FAICAR to IMP with the elimination of water.
Figure 2
Figure 2
Protomer structure of MthPurO/IMP. (A) Protomer of MthPurO/IMP complex shown in ribbon diagram with α-helices in blue and β-strands in green. MthPurO has a typical NTN hydrolase fold with an αββα structure. The IMP molecule is bound between two antiparallel β-sheets. The IMP molecule is represented by a ball-and-stick model with carbon in black, oxygen in red, nitrogen in blue, and phosphate in magenta. (B) Topology diagram of MthPurO. The first and the last residue numbers are labeled for each secondary structural element. The location of the active site is marked with a red asterisk.
Figure 3
Figure 3
Quaternary structure of MthPurO/IMP. Tetramer of MthPurO/IMP with each protomer shown in different color. The IMP molecules shown in ball-and-stick representation are bound in each protomer.
Figure 4
Figure 4
Stereoview of Fobs-Fcalc electron density around the active site of MthPurO in complex with (A) IMP and (B) AICAR contoured at 3.0 σ levels.
Figure 5
Figure 5
Surface representation of the MthPurO/IMP protomer. (A) Protomer surface in the same orientation as in Figure 2A. Strictly and strongly conserved residues within PurO sequences are mapped onto the molecular surface as red and yellow, respectively. The strictly conserved region coincides with a deep substrate binding pocket on the protein surface. (B) Close up view of the substrate binding pocket of MthPurO/IMP. The inosine moiety of the IMP sits deeply in the pocket shielded from the bulk solvent. Four charged residues, Arg5, Arg30, Glu104, and Asp106 are found in this region. (C) Multiple sequence alignment of PurOs. Strictly and strongly conserved residues within PurO sequences are shown in red and yellow boxes, respectively. The secondary structure of MthPurO is placed above the sequences. The residues that are involved in the purine base binding site (#), ribose binding (·), and phosphate binding (*) are marked below the sequences. All these active site residues are absolutely conserved in PurO sequences. METTH, Methanobacterium thermoautotrophicus; METJA, Methanocaldococcus jannaschii; METMP, Methanococcus maripaludis; HALN1, Halobacterium sp. NRC-1; METKA, Methanopyrus kandleri; HALMA, Haloarcula marismortui ATCC 43049; PYRKO, Thermococcus (Pyrococcus) kodakaraensis KOD1.
Figure 6
Figure 6
Active site of PurO with bound ligands. (A) Stereoview of the MthPurO/IMP active site. The IMP molecule and protein residues are represented by yellow and grey carbon atoms, respectively, with oxygen in red, nitrogen in blue, and phosphate in magenta. Hydrogen bonds are shown as broken lines. (B) Stereoview of the MthPurO/AICAR active site with the same atom colors as in (A).
Figure 7
Figure 7
Proposed mechanism A for the PurO-catalyzed cyclization of FAICAR. In mechanism A the reaction is initiated by ionization and tautomerization of the 4-carboxamide group of FAICAR 1 to the iminol 2/3. The tautomerization is assisted by an unusual pairing of tyrosine residues (Tyr20 and Tyr59) in which the environment of the Tyr20 hydroxyl group is entirely hydrophobic except for a strong hydrogen bond to the hydroxyl group of Tyr59. The tautomerization is further assisted by polarization of the carbonyl group by Arg30. Addition of the iminol nitrogen atom to the 5-formamide results in the tetrahedral intermediate 4. Tautomerization to 5 followed by loss of water results in IMP 6. R = ribose 5-phosphate.
Figure 8
Figure 8
Alternate mechanism B for the PurO-catalyzed cyclization of FAICAR. In mechanism B tautomerization of the 4-carboxamide of FAICAR to iminol occurs as in mechanism A. Tautomerization of the 4-formamide, assisted by Glu104, results in 7. A 6π electrocyclization reaction gives 5 and the product IMP 6 is generated by the same chemistry as in mechanism A. R = ribose 5-phosphate.
Figure 9
Figure 9
Active site comparison of MthPurO and PurH. The active sites are shown schematically in (A) MthPurO/IMP and (B) the IMP cyclohydrolase domain of human PurH/XMP. All key residues are different between PurO and PurH. In the PurH active site, most of the critical interactions between the protein and XMP are provided by backbone carbonyl atoms or nitrogen atoms. The Lys137′ from the other protomer completes the active site of PurH mediated by the water molecule. For the MthPurO/IMP active site, the hydrogen bonding distances are the average values for four protomers.
Figure 10
Figure 10
Active site comparison of MthPurO and GFP. The active sites are superimposed with the catalytically important residues and ligands shown: Arg30-Glu104-IMP in MthPurO (yellow carbon) and Arg96-Glu222-chromophore in GFP (green carbon). The hydrogen bonds are represented by broken lines. The relative positions of the arginine residues, glutamic acid residues, and ligands in the two proteins superimpose well despite their differences in sequence, structure, and function. Water molecules in GFP have been suggested as a proton shuttle.
SCHEME 1
SCHEME 1
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