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

Abstract

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.

Microbial Cell Factories for Green Production of Vitamins

“…Based on the riboswitch mechanism, mutations in the genes of thiamine pyrophosphate kinase activity (thiN) and thiamine-related transport proteins (YkoD and YuaJ) were introduced in B. subtilis TH95. It was recently reported that thiamine biosynthesis is strictly regulated by TPP riboswitches in bacteria/eukaryotes and transcriptional repressors in archaea (Hwang et al, 2017). E. coli has emerged as the preferred cell factory for TPP production after a riboswitch-based biosensors enabled the discovery of thiamine transporters, combined with overexpression of the native thiFSGHCE and thiD genes, which are closely related to Fe-S metabolism (Figure 1A and Table 1; Cardinale et al, 2017).…”

Vitamin B1 (Thiamine) Metabolism and Regulation in Archaea

“…ThiC (HMP-P synthase; EC 4.1.99.17) is the major enzyme used by bacteria [40,41], plant chloroplasts [42] and archaea [43] to synthesize the aminopyrimidine ring of thiamine (Figures 2-4).…”

“…ThiD homologs (IPR004399) are widespread in all domains of life, including organisms that only salvage HMP and do not synthesize thiamine de novo. Archaeal ThiD proteins are standalone or fused to a ThiN-type ThMP synthase domain (see later discussion) [43,53,54].…”

“…Although initially annotated as ribose-1,5-bisphosphate isomerases (R15Pi) based on indirect assay [77], archaeal Thi4 homologs are found to be distinct from archaeal R15Pi of the e2b2 family [78,79] and demonstrated to catalyze thiazole synthase activity [56] that is transcriptionally repressed when thiamine and THZ levels are sufficient [43] and is required for thiazole ring formation [57]. In vitro, yeast Thi4 operates by a suicide mechanism by mobilizing the sulfur of its active site cysteine (C205) to form ADP-thiazole from NAD and glycine [55].…”

Open Issues for Protein Function Assignment in Haloferax volcanii and Other Halophilic Archaea

“…Its genome has been sequenced and carefully annotated [ 1 , 10 , 11 ]. A plethora of biological aspects have been successfully tackled in this species, with examples including DNA replication [ 4 ]; cell division and cell shape [ 12 , 13 , 14 , 15 , 16 ]; metabolism [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]; protein secretion [ 26 , 27 , 28 , 29 ]; motility and biofilms [ 30 , 31 , 32 , 33 , 34 , 35 ]; mating [ 36 ]; signaling [ 37 ]; virus defense [ 38 ]; proteolysis [ 39 , 40 , 41 , 42 , 43 , 44 ]; posttranslational modification (N-glycosylation; SAMPylation) [ 45 , 46 , 47 , 48 , 49 , 50 ]; gene regulation [ 21 , 25 , 51 , 52 , 53 , 54 , 55 ]; microproteins [ 56 , 57 , 58 ] and small noncoding RNAs (sRNAs) […”