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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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3D-structural models of archaeal THI4 family proteins compared to the X-ray structure ofNeurospora 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.
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Fig2: 3D-structural models of archaeal THI4 family proteins compared to the X-ray structure ofNeurospora 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.

Mentions: To further compare THI4 family proteins, the 3D-stuctures of the HVO_0665, TK0434 and MA_2851 homologs were predicted by Phyre2-based fold-recognition and model building (see Methods for details). The resulting 3D-models were overlaid with the X-ray crystal structures of THI4p of S. cerevisiae (PDB: 3FPZ) and N. crassa (PDB: 3JSK) (Figure 2) to identify structurally conserved active site residues using Chimera 1.7 to visualize the models [21]. Conserved active site residues were identified based on analogy to residues shown to be important in ScTHI4p structure and activity [5] as well as residues bound to adenosine diphosphate 5-(beta-ethyl)-4-methyl-thiazole-2-carboxylic acid (AHZ) in the atomic structure of N. crassa THI4p. From this analysis, all three archaeal THI4 homologs were found to have close structural similarity to the yeast and fungal enzymes (Figure 2A-C). However, only the HvThi4 protein HVO_0665 (not TK0434 or MA_2851) was found to have residues analogous to those bound to AHZ in the atomic structure of N. crassa THI4p as well as the conserved catalytic cysteine of ScTHI4p required for sulfur mobilization to the thiazole ring (Figure 2B).Figure 2


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)

3D-structural models of archaeal THI4 family proteins compared to the X-ray structure ofNeurospora 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: 3D-structural models of archaeal THI4 family proteins compared to the X-ray structure ofNeurospora 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.
Mentions: To further compare THI4 family proteins, the 3D-stuctures of the HVO_0665, TK0434 and MA_2851 homologs were predicted by Phyre2-based fold-recognition and model building (see Methods for details). The resulting 3D-models were overlaid with the X-ray crystal structures of THI4p of S. cerevisiae (PDB: 3FPZ) and N. crassa (PDB: 3JSK) (Figure 2) to identify structurally conserved active site residues using Chimera 1.7 to visualize the models [21]. Conserved active site residues were identified based on analogy to residues shown to be important in ScTHI4p structure and activity [5] as well as residues bound to adenosine diphosphate 5-(beta-ethyl)-4-methyl-thiazole-2-carboxylic acid (AHZ) in the atomic structure of N. crassa THI4p. From this analysis, all three archaeal THI4 homologs were found to have close structural similarity to the yeast and fungal enzymes (Figure 2A-C). However, only the HvThi4 protein HVO_0665 (not TK0434 or MA_2851) was found to have residues analogous to those bound to AHZ in the atomic structure of N. crassa THI4p as well as the conserved catalytic cysteine of ScTHI4p required for sulfur mobilization to the thiazole ring (Figure 2B).Figure 2

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