Functional promiscuity of the COG0720 family

Abstract

The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH(4)), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G(+)) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins includes the 6-pyruvoyltetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologues, all members of the COG0720 family that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H(2)NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH(4) pathway, PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway, and PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, phylogenetic codistribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis and resulted in the identification of PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families extensive comparative genomic analysis beyond homology is required to correctly predict function.

Figure 1. Separation of COG0720 into six subfamilies by comparative genomic analysis

(A) Known or predicted roles of COG0720 proteins in GTP-derived metabolic pathways; (B) Physical clustering of the four PTPS sub-families (I–IV) with genes of folate, BH₄, Q or riboflavin synthesis pathways; Abbreviations and enzyme names described in main text.

Figure 2. Role of PTPS-I/III in both Q biosynthesis and THF biosynthesis

(A) Distribution of dual PTPS-I/III in both Q and THF biosynthesis in specific organisms; (B) Analysis by LC-MS/MS of Q content in bulk tRNA extracted from different strains showing the complementation of Q phenotype. Starting from the upper panel going down, it shows the UV trace of the digested bulk tRNA extracted from the isogenic wild type MG1655 pBAD24 (VDC3339), MG1655 ΔqueD pBAD24 (VDC3325), MG1655 ΔqueD pPTPS-I/III_Sa, and MG1655 ΔqueD pPTPS-II_Rn. The insets represent the extraction ion chromatograms for ions corresponding to 410 m/z; (C) Complementation of the dT auxotrophy phenotype of the E. coli ΔfolB strain by different COG0720 derivatives. Growth was monitored after 48 hours on LB plates containing 100 µg/mL Amp and supplemented when noted with 0.2% Ara or 80 µg/mL dT. Genome abbreviations: Sa: Synthrophus aciditrophicus, Cb: Clostridium botulinum, Ec: Escherichia coli, Rn: Rattus norvegicus

Figure 3. Genetic evidence for the possible functions of Archaeal COG0720

(A) Analysis by LC-MS/MS of G⁺ content in bulk tRNA extracted from different strains from the H. volcanii isogenic wild type (H26) upper panel and H26 ΔHVO_1718 (VDC3290) lower panel. (B) Genetic evidence that HVO_1282 (PTPS-IV) gene is not involved in folate or riboflavin biosynthesis. Growth of H. volcanii derivatives after 10 days growth on Hv –YPC plates with or without 80 µg/mL dT; 1- H. volcanii isogenic wild type (H26), 2- H26 ΔHVO_2182 and 3- H26 ΔfolE2; (C) Genetic evidence that HVO_1282 (PTPS-IV) gene is not involved in riboflavin biosynthesis. Growth of H. volcanii derivatives after 10 days growth on Hv –Mm plates with or without 20 µg/mL riboflavin; 1- H. volcanii isogenic wild type (H26), 2- H26 ΔHVO_1284 and 3- H26 Δ1282. (D) Genetic evidence that SSO2412 gene (PTPS-VI) is involved in folate biosynthesis. Complementation of dT auxotrophy phenotype of E. coli ΔfolB with SSO2412 cloned in pBAD24. Growth was monitored after 48 hours on LB plates containing 100 µg/mL Amp and supplemented when noted with 0.2% Ara or 80 µg/mL dT. Genome abbreviation: Ec: Escherichia coli.

Figure 4. LC-MS analysis of the E. coli QueD catalyzed reaction of 7,8-dihydroneopteirn monophosphate (H₂NMP)

(A) LC chromatogram of the reaction products monitored at an absorbance of 340 nm. Extracted ion chromatograms for ions corresponding to m/z (B) 210 (7,8-dihydropterin-6-carboxylic acid), (C) 208 (pterin-6-carboxylic acid), 166 (7,8-dihydropterin), and (E) 164 (pterin). The peak at ~12 min present in the absorbance chromatogram corresponds to an impurity present in the sample of H₂NMP. (F) Fluorescent HPLC Traces of (I.) GTP Cyclohydrolase IB (MJ0775) incubated with GTP showing the formation of neopterin cyclic phosphate (neopterin-cP); (II) MJ1272 incubated with GTP Cyclohydrolase 1A and GTP showing the conversion of neopterin-cP to pterin and 6-carboxypterin; and (III) MJ1272 incubated with sepiapterin showing the formation of 6-carboxypterin and pterin. The reaction products 6-carboxypterin and pterin were confirmed by GC-MS of their TMS-derivatives.”

Figure 5. Spatial comparisons of the active-site regions of PTPS-II from R. norvegicus, PTPS-I from P. aeruginosa and PTPS-III from P. falciparum

(A). Using Accelerys DS Vizualizer 2.5, R. norvegicus PTPS-II (black, PDB: 1B66) and the P. aeruginosa PTPS-I (grey, PDB: 2OBA) structures were superimposed with a rmsd 0.3942. (B) Close up of the three His residues coordinating the essential Zn²⁺ ion used as the reference points to show the relative occupation in space of the active-site nucleophile Cys42 in R. norvegicus PTPS-II (black) and the proposed nucleophile Cys24 in P. aeruginosa PTPS-I (grey). Distances of the respective nucleophilic centers (the S atom of Cys42 and Cys24) from the Zn²⁺ ion were measured as shown in the inset table. The distances from the O atom of C1OH and C2OH of the biopterin side chain were also measured and shown in the inset table. (C) Superimposition of the structures of PTPS-I (PDB: 2OBA, grey) and PTPS-III (PDB: 1Y13, black) was performed using the bioinformatics server FATCAT tool imbedded in PDB. The structure alignment has 116 equivalent positions with an optimum rmsd of 2.21 without twists. (D) Close up of the three His residues coordinating the essential Zn²⁺ ion used as the reference points to show the relative occupation in space of the active-site nucleophile Glu38 in P. falciparum PTPS-III (black) and the nucleophile Cys24 in P. aeruginosa PTPS-I (grey). Distances of the respective nucleophilic centres (the O atom of Glu38 and S from Cys24) from the Zn²⁺ ion were measured as shown in the inset table. The distances from the O atom of C1OH and C2OH of the biopterin side chain were also measured and shown in the table.

Figure 6

A condensed summary of the reactions involved in the mechanisms of PTPS-I, PTPS-II, and PTPS-III to generate their specific products.