[show abstract][hide abstract] 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.
ACS Chemical Biology 01/2012; 7(1):197-209. · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: GTP cyclohydrolase I (GCYH-I) is an essential Zn(2+)-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in approximately 25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn(2+)-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn(2+) starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn(2+)-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.
Journal of bacteriology 09/2009; 191(22):6936-49. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: GTP cyclohydrolase I (GCYH-I) is the first enzyme of the de novo tetrahydrofolate biosynthetic pathway present in bacteria, fungi, and plants, and encoded in Escherichia coli by the folE gene. It is also the first enzyme of the biopterin (BH4) pathway in Homo sapiens, where it is encoded by a homologous folE gene. A homology-based search of GCYH-I orthologs in all sequenced bacteria revealed a group of microbes, including several clinically important pathogens, that encoded all of the enzymes of the tetrahydrofolate biosynthesis pathway but GCYH-I, suggesting that an alternate family was present in these organisms. A prediction based on phylogenetic occurrence and physical clustering identified the COG1469 family as a potential candidate for this missing enzyme family. The GCYH-I activity of COG1469 family proteins from a variety of sources (Thermotoga maritima, Bacillus subtilis, Acinetobacter baylyi, and Neisseria gonorrhoeae) was experimentally verified in vivo and/or in vitro. Although there is no detectable sequence homology with the canonical GCYH-I, protein fold recognition based on sequence profiles, secondary structure, and solvation potential information suggests that, like GCYH-I proteins, COG1469 proteins are members of the tunnel-fold (T-fold) structural superfamily. This new GCYH-I family is found in approximately 20% of sequenced bacteria and is prevalent in Archaea, but the family is to this date absent in Eukarya.
Journal of Biological Chemistry 01/2007; 281(49):37586-93. · 4.65 Impact Factor