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

Hwang S, Cordova B, Chavarria N, Elbanna D, McHugh S, Rojas J, Pfeiffer F, Maupin-Furlow JA - BMC Microbiol. (2014)

Bottom Line: 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).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.Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA. sungminhwang@ufl.edu.

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.

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Related in: MedlinePlus

Haloferax volcaniiHVO_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.
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Fig1: Haloferax volcaniiHVO_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.

Mentions: To determine the metabolic potential of the archaeal members of the THI4 protein family (IPR002922) in thiamine biosynthesis, archaeal homologs were compared to THI4p of yeast (Saccharomyces cerevisiae, ScTHI4) and plant (Arabidopsis thaliana, AtTHI4) by multiple amino acid sequence alignment and cluster analysis (Figure 1A-B). By these comparisons, the majority of archaeal THI4 homologs were found to have a histidine residue conserved in the position of the active site cysteine residue (Cys205) of the yeast THI4p, with some even having a proline residue. Archaeal Thi4 ‘histidine-containing’ homologs from methanogenic archaea (MJ0601 and MA_2851) are implicated in R15P isomerase reactions not associated with thiamine metabolism to provide ribulose-bisphosphate as a substrate of archaeal ribulose-bisphosphate carboxylase [6]. In a subsequent study, archaeal R15P isomerases were put into a wider context of CO2 fixation via the AMP pathway [7]. Phylogenetic profile analysis as well as in vivo and in vitro studies assigned R15P isomerase activity to TK0185, a protein unrelated to THI4p [7,22]. By contrast, the THI4p homolog of T. kododaraensis (TkThi4, TK0434) did not show any R15P isomerase activity by in vitro analysis [7]. Even though the results of these earlier studies are controversial [6,7], none implicates a THI4p homolog to be involved in thiamine biosynthesis in an archaeon.Figure 1


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

Hwang S, Cordova B, Chavarria N, Elbanna D, McHugh S, Rojas J, Pfeiffer F, Maupin-Furlow JA - BMC Microbiol. (2014)

Haloferax volcaniiHVO_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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4215014&req=5

Fig1: Haloferax volcaniiHVO_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.
Mentions: To determine the metabolic potential of the archaeal members of the THI4 protein family (IPR002922) in thiamine biosynthesis, archaeal homologs were compared to THI4p of yeast (Saccharomyces cerevisiae, ScTHI4) and plant (Arabidopsis thaliana, AtTHI4) by multiple amino acid sequence alignment and cluster analysis (Figure 1A-B). By these comparisons, the majority of archaeal THI4 homologs were found to have a histidine residue conserved in the position of the active site cysteine residue (Cys205) of the yeast THI4p, with some even having a proline residue. Archaeal Thi4 ‘histidine-containing’ homologs from methanogenic archaea (MJ0601 and MA_2851) are implicated in R15P isomerase reactions not associated with thiamine metabolism to provide ribulose-bisphosphate as a substrate of archaeal ribulose-bisphosphate carboxylase [6]. In a subsequent study, archaeal R15P isomerases were put into a wider context of CO2 fixation via the AMP pathway [7]. Phylogenetic profile analysis as well as in vivo and in vitro studies assigned R15P isomerase activity to TK0185, a protein unrelated to THI4p [7,22]. By contrast, the THI4p homolog of T. kododaraensis (TkThi4, TK0434) did not show any R15P isomerase activity by in vitro analysis [7]. Even though the results of these earlier studies are controversial [6,7], none implicates a THI4p homolog to be involved in thiamine biosynthesis in an archaeon.Figure 1

Bottom Line: 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).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.Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA. sungminhwang@ufl.edu.

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.

Show MeSH
Related in: MedlinePlus