Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii

Background Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms. Results Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant. Conclusions Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0260-0) contains supplementary material, which is available to authorized users.

Thiamine salvage. Microbes have evolved transporters and kinases to uptake and salvage thiamine derivatives present in the environment (Fig. 1C). In bacteria, an ABC-type transporter (ThiBPQ) is used for the uptake of thiamine and TPP and appears conserved in Hfx. volcanii. The putative transmembrane protein HVO_0023 of the UPF0118 superfamily may associate with this ABC-type thiamine transporter based on genome neighborhood linkage. Hfx. volcanii is also predicted to uptake thiamine precursors by a symport mechanism based on coding sequence overlap of HVO_B0379 (PtuP2, a Na + /solute symporter homolog) with HVO_B0380 (TenA2, a homolog of bacterial TenA and yeast THI20 C-terminal domain thiaminase II enzymes). Thiaminase II cleaves thiamine related compounds including those generated by YlmB-mediated deformylation to generate hydroxymethylpyrimidine (HMP). HMP is successively phosphorylated through a series of ThiD mediated kinase reactions to synthesize HMP-PP. Hfx. volcanii has homologs to all of these enzymes (TenA, YlmB and ThiD) suggesting it can synthesize HMP-PP by a salvage pathway. Hfx. volcanii also appears to salvage HET-P through phosphorylation of THZ based on identification of the ThiM homolog HVO_2667. Thiamine pyrophosphokinase (TPK) enzymes of the IPR006282 family that convert thiamine to TPP were restricted to bacteria and eukaryotes with no homologs identified in Hfx. volcanii or other archaea.

Sulfur activation for the thiazole ring.
In the bacterial pathway, sulfur is provided for ThiG in an activated form, as thiocarboxylate on the C-terminal glycine of the carrier protein ThiS. Generation of this thiocarboxlate starts with activation of ThiS by adenylation, which is catalyzed by ThiF. The adenylate is then exchanged against a sulfur atom provided by ThiI. Homologs for all of these proteins are identified in Hfx. volcanii. ThiS has a ubiquitin-fold, and its Hfx. volcanii structural homologs (SAMP1, HVO_2619; SAMP2, HVO_0202; SAMP3, HVO_2177 with a corrected start codon to result in a 92 aa protein) were shown to be covalently attached to target proteins in a process called sampylation [7,8]. SAMP1 and SAMP2 were also shown to be involved in sulfur chemistry, SAMP1 participating in biosynthesis of molybdopterin while SAMP2 participates in thiolation of tRNA [9]. Hfx. volcanii has only a single E1-type enzyme (UbaA, HVO_0558) which belongs to the ThiF/MoeB/HesA family and adenylates all three SAMPs based on its requirement for SAMP function [8,9]. Thus, we have the rare opportunity to determine if any of the SAMPs are involved in sulfur chemistry of thiamine biosynthesis by analyzing a ΔubaA strain (this study).
An eventual involvement of the This-ThiF homologs, SAMP(1-3)-UbaA, would require sulfur transfer from ThiI (HVO_1651). However, Salmonella enterica ThiI provides sulfur for thiamine biosynthesis via its rhodanese domain, a domain also occurring in the E. coli ortholog [10]. This rhodanese domain is found in a minority of the ThiI homologs and is missing from HVO_1651, making involvement on HVO_1651 in thiamine biosynthesis rather unlikely. In addition to thiamine biosynthesis, S. enterica ThiI is also involved in thiolation of tRNA, a function which requires only the two N-terminal domains [10]. Thus, nearly all of the proteins named "thiamine biosynthesis protein ThiI" in the databases are concluded to be completely unrelated to thiamine biosynthesis but instead are involved in generation of the modified tRNA base 4-thiouridine [11]. Similarly to the methanogen homolog MMP1354, this is also the likely function of HVO_1651 as we find it is not required for growth of Hfx. volcanii in the absence of thiamine (data not shown).

Supplementary Tables
Suppl . Table S1. Haloferax volcanii DS2 gene homologs of thiamine (vitamin B1) metabolism and transport a,b .  c While UbaA shares 39% amino acid identity (over a query coverage 90%) with E. coli ThiF, UbaA is not required for thiamine biosynthesis (this study) and instead functions with the ubiquitin-fold SAMPs in the formation of ubiquitin-like isopeptide bonds, the thiolation of tRNA, and the biosynthesis of molybdopterin (MPT) [8,9]. d HVO_1651 is related to ThiI but devoid of the rhodanese domain (RHD). HVO_1651 is not required for thiamine biosynthesis (this study) and likely functions in tRNA modification based on analogy to methanogens [18]. e Hfx. volcanii SAMPs are Ub-fold proteins structurally related to ThiS [28][29][30] and function with UbaA in sulfur transfer and protein modification [8,9,27]. However, SAMPs do not appear to be linked with thiamine metabolism based on analysis of UbaA (this study). Suppl.

Select bacteria
Q9WZP4_ Thermotoga maritima VMMTG---LHVDPLTVEAKF a Residues in analogous position to conserved active site cysteine (Cys205) of ScTHI4p are highlighted (i.e., cysteine residues in red, histidine residues in black, and proline residues in blue). Hfx. volcanii HVO_0665 is the THI4p homolog of this study. MJ0601 and MA_2851 are described as a D-ribose-1,5-bisphosphate isomerases [53]. TK0434 is annotated as a putative ribose 1,5-bisphosphate isomerase but was demonstrated to lack this activity in vitro [54]. Note that select species of methanogens and pyrococci have two THI4 homologs including one with a conserved active site cysteine and another with a histidine in this position. UniProtKB/Swiss-Prot numbers are listed for each protein sequence. Gaps introduced to optimize multiple amino acid sequence alignment are indicated by -.    Table S1 include: HVO_0109 (D4GYV5, SufS-type cysteine desulfurase) [55]; HVO_B0001 (D4GP02, Orc1-type DNA replication protein); HVO_B0382 (D4GQ28, TATAbox-binding protein 3 or Tbp3). Hfx. volcanii homologs of bacterial glycine oxidase (ThiO), thiazole synthase (ThiG), thiamine phyrophosphokinase (TPK) and thiazole tautomerase (TenI) in addition to yeast HMP-P synthase (THI5) and thiamine pyrophosphokinase (THI80) were not detected. Conversion of ADT to THZ-P is predicted to be catalyzed by a NUDIX hydrolase domain enzyme that has yet to be identified [56]. Members of the RHD (IPR001763) and NUDIX hydrolase domain (IPR000086) are common in Hfx. volcanii. HVO_1651 is related to bacterial ThiI but devoid of the rhodanese-like domain (RHD), which alone mediates the catalytic function of ThiI in thiamine biosynthesis [10,11]