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

Abstract

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.

Figure 1

Haloferax volcanii HVO_0665 (HvThi4) is related to members of the THI4 protein family (IPR002922). (A) Multiple amino acid sequence alignment of THI4 homologs including Hfx. volcanii HVO_0665 (HvThi4), Saccharomyces cerevisiae ScTHI4, Arabidopsis thaliana AtTHI4, Thermotoga maritima Tmari_0788, Methanosarcina acetivorans MA_2851, Methanocaldococcus jannaschii MJ0601 and Thermococcus kodakarensis TK0434. Identical and functionally similar amino acid residues are highlighted in black and grey, respectively, with residues conserved with the ScTHI4 Cys205 active site highlighted in red. α helices and β sheets predicted for HVO_0665 by Phyre2-based homology modeling are indicated above the alignment. (B) Cluster analysis of HvThi4 with members of the THI4 protein family. HVO_0665 (HvThi4) of this study is indicated by a circle (●). M. acetivorans MA_2851 and M. jannaschii MJ0601 described as D-ribose-1,5-bisphosphate isomerases and the associated T. kodakarensis TK0434 demonstrated to lack this activity are indicated by squares (■). S. cerevisiae and A. thaliana THI4 enzymes of thiamine biosynthesis are indicated by triangles (▲). Cluster of archaeal THI4 homologs with a conserved active site cysteine residue analogous to ScTHI4 Cys205 are shaded in blue and include uncharacterized proteins of halophilic archaea, Thaumarchaeota, Aeropyrum, and select methanogens and pyrococci. Three letter genus abbreviations are used as proposed by the Subcommittee on the taxonomy of the family Halobacteriaceae. N- and C-termini were trimmed for protein alignments. UniProtKB accession numbers associated with protein sequences are listed in supplemental information.

Figure 2

3D-structural models of archaeal THI4 family proteins compared to the X-ray structure of Neurospora crassa (Nc) THI4p (PDB: 3JSK). Proteins are represented in ribbon diagram including HVO_0665 (HvThi4, dark blue), MA_2851 (cyan), TK0434 (purple) and NcTHI4p (light brown), with the latter in octameric (A) and monomeric (B) configuration. For clarity in panel B, N- and C-terminal amino acid extensions of HvThi4 (residues 1–9 and 298–307) and NcTHI4p (residues 35–57) are hidden. (C) NcTHI4p residues bound or in close proximity to adenosine diphosphate 5-(beta-ethyl)-4-methyl-thiazole-2-carboxylic acid (AHZ) are indicated with structurally analogous residues of HvThi4 highlighted (where .a and .b indicate residues of chains a and b at the dimer interface). The conserved catalytic cysteine residue of NcTHI4p (Cys232) that is essential for thiamine biosynthesis is in the sulfur minus 2,3-didehydroalanine (DHA) form and is structurally analogous to HvThi4 Cys165 as indicated in pink.

Figure 3

The Haloferax volcanii thi4 gene and its in-frame deletion. (A) Schematic representation of the thi4 gene on the genome of Hfx. volcanii DS2. HVO_0662 encodes a ThiN homolog with a predicted N-terminal helix-turn-helix (HTH) DNA binding domain. The PCR primer pairs (P1/P2 and P3/P4) used to generate the thi4 gene deletion and the PCR primer pairs (P5/P6 and P7/P8) used to screen for the thi4 gene deletion are indicated. (B) PCR products generated for the Δthi4 (Δhvo_0665) mutant and parent (H26, wt) strains using primer pairs P5/P6 and P7/P8 as indicated. Size reduction with primer pair P7/P8 and the absence of a signal with primer pair P5/P6 confirm the deletion in the Δthi4 strain.

Figure 4

The Haloferax volcanii THI4 homolog is required for growth in the absence of thiamine and the thiazole moiety 4-methyl-5-(β-hydroxyethyl)thiazole (THZ). Hfx. volcanii strains including H26 parent (wt), HM1052 (ΔubaA), NC1011 (Δthi4), NC1011-pJAM202c (Δthi4 + empty vector), NC1011-pJAM2821 (Δthi4 + HvThi4), and NC1011-pJAM2822 (Δthi4 + HvThi4 C165A) were grown in GMM supplemented with and without thiamine or THZ as indicated and described in the Materials and Methods. First round (A) and successive growth curves (B and C) are presented with the mean ± standard deviations shown.

Figure 5

The THI4 homolog (HvThi4) and its C165A variant are synthesized with C-terminal StrepII tags as stable derivatives in Haloferax volcanii . Production of the HvThi4-StrepII and HvThi4 C165A-StrepII proteins in the trans complemented strains (NC1011-pJAM2821 and -pJAM2822, respectively) was detected by anti-StrepII Western blotting (0.065 OD₆₀₀ units of cells per lane). Equivalent protein loading was assessed by staining blots with Ponceau Red S (not shown) and parallel SDS-PAGE gels with Coomassie Blue R-250 (CB stain, 37–50 kDa range of gel shown).

Figure 6

The pathway for de novo biosynthesis of thiamine in the archaeon Haloferax volcanii is an apparent hybrid of bacterial and yeast pathways. Yeast and bacterial like steps are shaded by purple and blue, respectively, with associated enzymes of thiamine biosynthetic indicated in text with similar color coding. Hfx. volcanii homologs are indicated by gene locus tag in green. Question mark (?) designated enzyme is yet unassigned. THI4-SH specifies the catalytic cysteine side chain. THI4-C = CH indicates the dehydroalanine form of the enzyme after sulfur transfer. The sulfur atom associated with formation of the thiazole ring is highlighted in red. In the Hfx. volcanii model for thiamine biosynthesis, the thiazole moiety of thiamine (4-methyl-5-(β-hydroxyethyl)thiazole phosphate; HET-P or THZ-P) is generated by a yeast-like mechanism. HvThi4 (HVO_0665) converts nicotinamide adenine dinucleotide (NAD) and glycine to ADP-thiazole (ADT), which predicted to be hydrolyzed to THZ-P by a yet to be identified NUDIX-type hydrolase [29]. The remaining steps of thiamine biosynthesis are related to bacterial systems. In particular, the PurM-like AIR synthetase HVO_1557 is predicted to form 5-amino-1-(5-phospho-D-ribosyl)imidazole (synonym 5-aminoimidazole ribotide; AIR), which serves as a substrate for a ThiC-like S-adenosyl-methionine (SAM)-dependent HMP-P synthase (HVO_2154) in the generation of 4-amino-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P). Once generated, HMP-P is phosphorylated by a bacterial ThiD-type HMP-P kinase (HVO_2666) which is also conserved in the N-terminal domains (Ntd) of yeast THI20 and THI21. Thiamine-phosphate synthase homologs of the ThiE-type (HVO_2668) and ThiN-type (HVO_0662) are predicted to condense THZ-P with HMP-PP to form thiamine monophosphate (TMP). TMP is then phosphorylated to TPP by a proposed bacterial ThiL-type thiamine-monophosphate kinase (HVO_1861).