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

GTP cyclohydrolase I (GCYH-I) is an essential Zn²⁺-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 ∼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 Zn²⁺-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn²⁺ 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, Zn²⁺-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.The Zn²⁺-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. ​(Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. ​(Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn²⁺ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH₄) pathway (Fig. ​(Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table ​Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).

TABLE 1.

Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteria^a

Organismc	Presence of:
	folE	folE2
Enterobacteria
Escherichia coli	+	−
Salmonella typhimurium	+	−
Yersinia pestis	+	−
Klebsiella pneumoniaeb	+	+a
Serratia marcescens	+	+a
Erwinia carotovora	+	−
Photorhabdus luminescens	+	−
Proteus mirabilis	+	−
Gammaproteobacteria
Vibrio cholerae	+	−
Acinetobacter sp. strain ADP1	+	+a
Pseudomonas aeruginosa	+	+a
Pseudomonas entomophila L48	+	+a
Pseudomonas fluorescens Pf-5	+	+a
Pseudomonas syringae	+	+a
Pseudomonas putida	+	+a
Hahella chejuensis KCTC 2396	+	+a
Chromohalobacter salexigens DSM 3043	+	+a
Methylococcus capsulatus	+	+a
Xanthomonas axonopodis	+	+a
Xanthomonas campestris	+	+a
Xylella fastidiosa	+	+a
Idiomarina loihiensis	−	+
Colwellia psychrerythraea	+	+
Pseudoalteromonas atlantica T6c	+	+a
Pseudoalteromonas haloplanktis TAC125	+	+
Alteromonas macleodi	+	−
Nitrosococcus oceani	+	+
Legionella pneumophila	+	−
Francisella tularensis	+	−
Betaproteobacteria
Chromobacterium violaceum	+	−
Neisseria gonorrhoeae	−	+
Burkholderia cepacia {"type":"entrez-nucleotide","attrs":{"text":"R18194","term_id":"771804","term_text":"R18194"}}R18194	+	+
Burkholderia cenocepacia AU 1054	+	+
Burkholderia xenovorans	+	−
Burkholderia mallei	+	−
Bordetella pertussis	−	+
Ralstonia eutropha JMP134	+	−
Ralstonia metallidurans	+	+
Ralstonia solanacearum	+	−
Methylobacillus flagellatus	−	+
Nitrosomonas europaea	−	+
Azoarcus sp.	+	+
Bacilli/Clostridia
Bacillus subtilisd	+	+
Bacillus licheniformis	+	+
Bacillus cereus	+	−
Bacillus halodurans	+	+
Bacillus clausii	+	−
Geobacillus kaustophilus	+	−
Oceanobacillus iheyensis	−	+
Staphylococcus aureus	−	+

Open in a separate window^aGenes that are preceded by candidate Zur binding sites.^bZur-regulated cluster is on the virulence plasmid pLVPK.^cExamples of organisms with no folE genes are in boldface type.^dZn-dependent regulation of B. subtilis folE2 by Zur was experimentally verified (17).Expression of the Bacillus subtilis folE2 gene, yciA, is controlled by the Zn²⁺-dependent Zur repressor and is upregulated under Zn²⁺-limiting conditions (17). This led us to propose that the GCYH-IB family utilizes a metal other than Zn²⁺ to allow growth in Zn²⁺-limiting environments, a hypothesis strengthened by the observation that an archaeal ortholog from Methanocaldococcus jannaschii has recently been shown to be Fe²⁺ dependent (22). To test this hypothesis, we investigated the physiological role of GCYH-IB in B. subtilis, an organism that contains both isozymes, as well as the metal dependence of B. subtilis GCYH-IB in vitro. To gain a structural understanding of the metal dependence of GCYH-IB, we determined high-resolution crystal structures of Zn²⁺- and Mn²⁺-bound forms of the N. gonorrhoeae ortholog. Notably, although the GCYH-IA and -IB enzymes belong to the tunneling-fold (T-fold) superfamily, there are significant differences in their global and active-site architecture. These studies shed light on the physiological significance of the alternative folate biosynthesis isozymes in bacteria exposed to various metal environments, and offer a structural understanding of the differential metal dependence of GCYH-IA and -IB.