Enzymatic and structural characterization of an archaeal thiamin phosphate synthase

Hayashi, Maria; Kobayashi, Kazuya; Esaki, Hiroyoshi; Konno, Hiroyuki; Akaji, Kenichi; Tazuya, Keiko; Yamada, Kazuko; Nakabayashi, Toshikatsu; Nosaka, Kazuto

doi:10.1016/j.bbapap.2014.02.017

Cited by 12 publications

(16 citation statements)

References 15 publications

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“…1B and 4B), whereas ThiN group II proteins are active, based on results of growth assays (Fig. 4B) and data from previous studies (19,28). This finding is surprising in that the ThiN domains of group I and II proteins have relatively high sequence similarity (e.g., 39% in N. magadii), including conserved residues predicted to function in catalytic activity (e.g., Glu297, Arg321, and His342 in NmThiDN).…”

Section: Discussionmentioning

confidence: 47%

“…While the active-site residues of the ThiN-like TPS enzyme reported for the condensation of THZ-P and HMP-PP were conserved in HvThiN (19), our results clearly demonstrated that the product of the thiN gene was not required for catalyzing thiamine biosynthesis and could not compensate for the loss of thiE function in H. volcanii. HvThiN is naturally fused to an N-terminal HTH DNA binding domain predicted by homology modeling to be structurally related to XRE-type (XRE_HTH) transcriptional repressors of bacteriophages cro, CI, and lambda ( Fig.…”

mentioning

confidence: 51%

“…This finding is surprising in that the ThiN domains of group I and II proteins have relatively high sequence similarity (e.g., 39% in N. magadii), including conserved residues predicted to function in catalytic activity (e.g., Glu297, Arg321, and His342 in NmThiDN). Previous work revealed that the conserved His342 and Arg321 residues are important for ThiN group II activity as determined by in vitro assays (19). However, this feature alone does not explain why ThiN group I proteins are catalytically inactive, since these two residues are conserved regardless of classification into group I or II (Fig.…”

Section: Discussionmentioning

confidence: 82%

“…A previous study using site-directed mutagenesis and in vitro assays revealed that conserved Arg320 and His341 residues are required for the TPS activity of Pyrobaculum calidifontis ThiDN (analogous to Arg321 and His342 of N. magadii ThiDN, with the latter numbering being used in this study) (19). However, the current understanding of residues critical for the ThiN domain-mediated condensation of THZ-P and HMP-PP to TMP is limited since the two residues (Arg321 and H342) are conserved in ThiN domains regardless of their classification into group I or II, with the ThiR type (group I) being found to be catalytically inactive by our study (Fig.…”

Section: Thin Domain Functionmentioning

confidence: 99%

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ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis

et al. 2017

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Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax volcanii, one of the representative archaea, uses a eukaryote-like Thi4 (thiamine thiazole synthase) for the production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterium-like ThiC (2-methyl-4-amino-5-hydroxymethylpyrimidine [HMP] phosphate synthase) branch. Here we found that archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor that harbored an N-terminal helix-turn-helix (HTH) DNA binding domain and C-terminal ThiN (TMP synthase) domain was identified. In the presence of thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found to be catalytically inactive in compensating for the loss of ThiE (TMP synthase) function. In contrast, bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP phosphate [HMP-P] kinase) domain, was found to be interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended ␣-helix near the active-site His of the ThiN domain was found to be important for ThiDN catalytic activity, whereas the corresponding Met residue was absent and the ␣-helix was shorter in ThiR homologs. Thus, we provide new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor, ThiR, and not by a riboswitch.IMPORTANCE Thiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes, including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryote-and bacterium-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find that ThiN is a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveals that archaeal thiamine biosynthesis is regulated by a ThiN domain transcriptional repressor, ThiR, and not by a riboswitch.

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Section: Discussionmentioning

confidence: 47%

mentioning

confidence: 51%

Section: Discussionmentioning

confidence: 82%

Section: Thin Domain Functionmentioning

confidence: 99%

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ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis

et al. 2017

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“…The de novo pathway of thiamin biosynthesis involves the independent formation of HMP pyrophosphate (HMP-PP) and HET phosphate (HET-P), as well as their subsequent condensation to form TP by thiamin phosphate synthase common in all organisms (2)(3)(4). In eubacteria and eukaryotes, ThiE protein and its orthologs play the role of thiamin phosphate synthase, whereas ThiN protein acts as the catalyst of the same reaction in most archaea (5,6). Then the final product TPP is synthesized from TP in the de novo pathway (Fig.…”

mentioning

confidence: 99%

Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon <i>Pyrobaculum calidifontis</i>

Hayashi

Nosaka

2015

J Nutr Sci Vitaminol

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Summary Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thiazole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thiamin phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphokinase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidifontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P. calidifontis. Furthermore, the results obtained by site-directed mutagenesis suggest that the Ser196 of ThiL protein plays a pivotal role in the catalytic process. Key Words thiamin phosphate kinase, thiamin pyrophosphate, thiamin synthesis, ThiL, Pyrobaculum calidifontis Thiamin, also known as vitamin B1, occurs in cells as free thiamin and as its phosphate esters, thiamin phosphate (TP), thiamin pyrophosphate (TPP) and thiamin triphosphate (1). TPP is a cofactor for many enzymes indispensable for glucose and energy metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and transketolase. Thiamin consists of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine, HMP) and 4-methyl-5-bhydroxyethylthiazole (hydroxyethylthiazole, HET). The de novo pathway of thiamin biosynthesis involves the independent formation of HMP pyrophosphate (HMP-PP) and HET phosphate (HET-P), as well as their subsequent condensation to form TP by thiamin phosphate synthase common in all organisms (2-4). In eubacteria and eukaryotes, ThiE protein and its orthologs play the role of thiamin phosphate synthase, whereas ThiN protein acts as the catalyst of the same reaction in most archaea (5, 6). Then the final product TPP is synthesized from TP in the de novo pathway (Fig. 1). Many eubacterial cells synthesize TPP by the phosphorylation of TP in the presence of ATP and Mg 21 by thiamin phosphate kinase (TP kinase, ThiL, EC 2.7.4.16) (7). In eukaryotic cells and some eubacterial cells, this final step occurs in two stages: hydrolysis ...

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Panguiarchaeum symbiosum, a potential hyperthermophilic symbiont in the TACK superphylum

Rao

et al. 2023

Cell Reports

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Enzymatic and structural characterization of an archaeal thiamin phosphate synthase

Cited by 12 publications

References 15 publications

ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis

ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis

Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon <i>Pyrobaculum calidifontis</i>

Panguiarchaeum symbiosum, a potential hyperthermophilic symbiont in the TACK superphylum

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