Thiamin Biosynthesis - still yielding fascinating biological chemistry

Abstract

This paper will describe the biosynthesis of the thiamin thiazole in Bacillus subtilis and Saccharomyces cerevisiae. The two pathways are quite different: in B. subtilis, the thiazole is formed by an oxidative condensation of glycine, deoxy-D-xylulose- 5-phosphate and a protein thiocarboxylate, while in S. cerevisiae the thiazole is assembled from glycine, NAD and Cys205 of the thiazole synthase.

The major thiamin biosynthetic pathway in bacteria is outlined in Figure 1.(1, 2) In this pathway, glycine 3 undergoes an oxidative condensation with deoxy-D-xylulose- 5-phosphate 5 and ThiS-thiocarboxylate 6, to give the thiazole tautomer 7, which then aromatizes to form carboxythiazole 8.(3, 4) The thiamin pyrimidine 15 is formed by a remarkable rearrangement of AIR 14, an intermediate on the purine pathway.(5) Coupling the thiazole and the pyrimidine, with concomitant decarboxylation, yields thiamin phosphate 2.(6, 7) A final phosphorylation gives thiamin pyrophosphate 1, the biochemically active form of the cofactor.(8)

Figure 1.

The bacterial thiamin biosynthetic pathway.

Our understanding of this biosynthetic pathway is now at an advanced stage. All the biosynthetic genes have been identified and cloned, all of the enzymes have been overexpressed, reconstituted and structurally characterized and mechanisms for all of the biosynthetic reactions, except for the pyrimidine synthase (ThiC) are reasonably clear (1, 5). The entire biosynthetic pathway has been fully reconstituted using pure enzymes. In this lecture, I will describe the biosynthesis of the thiamin thiazole in B. subtilis and compare this pathway with the very different thiazole biosynthesis recently elucidated in S. cerevisiae.

Thiamin thiazole biosynthesis in B. subtilis

Each of the steps involved in the assembly of the thiamin thiazole in bacteria will be described in the following sections.

Glycine Oxidation

The ThiO gene product encodes a flavin-dependent glycine oxidase that catalyzes the oxidation of glycine 3 to the glycine imine 4 (9). In the absence of the other thiazole biosynthetic enzymes, the glycine imine is hydrolyzed to glyoxal.

The structure of this enzyme, with N-acetyl glycine bound at the active site, has been determined (PDB code = 1NG3). This structure and studies with substrate analogs are consistent with a hydride transfer mechanism for glycine oxidation (9).

In anaerobes, the glycine imine is formed from tyrosine in a reaction catalyzed by ThiH, a radical SAM enzyme (10–12).

ThiS-thiocarboxylate Formation

The chemistry involved in the formation of ThiS-thiocarboxylate is outlined in Figure 1. Activation of ThiS-COOH 9, at its carboxy terminus, by adenylation, gives 10 which then acylates the IscS persulfide to give 13. Reduction of 13, by DTT in the reconstitution reaction mixture, gives the ThiS thiocarboxylate 6 (13, 14). The biochemical reduction of 13 is not yet understood. In some bacteria, an additional protein (ThiI) mediates the sulfur transfer to 10.(15) ThiS-thiocarboxylate 6 can also be efficiently synthesized by treating intein-activated ThiS-COOH with ammonium sulfide (16).

The structures of the ThiF/ThiS complex and the ThiF/ATP complex have been determined (17, 18) (PDB codes = 1ZUD and 1ZFN). The IscS protein probably does not form a specific complex with ThiS/ThiF because all four IscS paralogs in B. subtilis are competent persulfide donors. (14, 19)

Protein thiocarboxylates as sulfide carriers in other biosynthetic pathways

Protein thiocarboxylates have now been found to play a role as sulfide carriers in several other biosynthetic pathways and sequence analysis suggests that this strategy may be quite general (Figure 2).

Figure 2.

Four additional examples of protein-thiocarboxylate-dependent biosynthetic pathways. A) Molybdopterin biosynthesis in bacteria, B) Cysteine biosynthesis in Mycobacterium tuberculosis, C) Homocysteine biosynthesis in Wolinella succinogenes, D) Thioquinolobactin biosynthesis in Pseudomonas fluorescens.

In molybdopterin biosynthesis, MoaE catalyzes the transfer of sulfide from MoaD-thiocarboxylate to give 19 (Figure 2A)(20, 21). A protein thiocarboxylate dependent cysteine biosynthetic pathway has been found in M. tuberculosis (Figure 2B). In this pathway, CysM thiocarboxylate reacts with phosphoserine 21 in a PLP-mediated reaction to form thioester 22. This then undergoes an N/S acyl shift to give 23 followed by release of cysteine in a hydrolysis reaction catalyzed by the Mec protease (22–26). A closely related pathway for the biosynthesis of homocysteine was discovered in Wolinella succinogenes (Figure 2C). In this pathway HcyS-thiocarboxylate 25 adds to O-acetyl homoserine 26 to give thioester 27. An N/S acyl shift to give 28 followed by HcyD-catalyzed amide hydrolysis generates homocysteine 30 (27, 28). A fourth example is found in the biosynthesis of the siderophore thioquinolobactin 34 (Figure 2D). In this pathway, QbsE thiocarboxylate forms a mixed thioanhydride 33 with quinolobactin 31. Hydrolysis of 33 generates the siderophore 34.(29, 30) A reagent for the sensitive detection of protein thiocarboxylates in a proteome, that uses a click reaction between the protein thiocarboxylate and a fluorophore-tagged sulfonyl azide, has been described. (31)

Formation of the thiazole tautomer 7

The bacterial thiazole synthase catalyzes the condensation of DXP (5), ThiS-COSH 6 and the glycine imine 4 to form the thiazole tautomer 7 (Figure 1)(3). A mechanistic proposal for this reaction is outlined in Figure 3. In this mechanism, DXP 5 forms an imine with Lysine 96 of the thiazole synthase. Tautomerization to 38 followed by thiocarboxylate addition gives 39. An O/S acyl shift followed by loss of water generates thioketone 41. Tautomerization of 41, followed by loss of ThiS-COOH generates 43. Addition of the glycine imine 4 followed by transimination gives the thiazole tautomer 7.

Figure 3.

Mechanistic proposal for the formation of the thiazole tautomer 7.

In support of this mechanism, enzyme-catalyzed exchange of the DXP carbonyl oxygen has been observed and the DXP/K96 imine has been trapped by borohydride reduction and characterized by MS analysis. Intermediate 37 is supported by the observation of enzyme-catalyzed exchange of the C3 proton of DXP. The unanticipated O/S acyl shift to give 40 is supported by the observation of oxygen incorporation from DXP and not the buffer into the nascent ThiS-COOH. Thioenol 42 has also been trapped and characterized by MS analysis and the final product 7 has been fully characterized by spectroscopic analysis (3, 14).

The structure of the ThiG/ThiS complex, with phosphate bound at the active site, has been determined (PDB code = 1TYG). In this structure the phosphate and Lys96 define the DXP binding site, which suggests that Glu98 and Asp182 are also likely to play a role in the catalysis of thiazole formation (32).

Thiazole tautomerase

The thiazole tautomer 7 is surprisingly stable and the aromatization reaction to produce the thiazole 8 requires enzymatic catalysis. In B. subtilis, the TenI protein has recently been identified as the thiazole tautomerase.

The structure of the enzyme product 8 complex has been determined (PDB code = 3QH2). A model of the enzyme substrate complex generated from this structure suggests that His122 mediates the deprotonation at C2 and that the substrate phosphate group functions as the proton donor for the exocyclic double bond protonation (4). TenI shows high sequence similarity to thiamin phosphate synthase and the two enzymes are frequently incorrectly assigned in genome annotation.

Thiamin thiazole biosynthesis in S. cerevisiae

The thiamin biosynthetic pathway in S. cerevisiae is outlined in Figure 4.(33) The biosynthesis of the thiazole and the pyrimidine heterocycles (5 &10) occurs by very different chemistry from that used for the bacterial biosynthesis. Labeling studies have demonstrated that the thiazole is formed from an unidentified C5 carbohydrate, glycine 3 and cysteine 11(34–36) and that the pyrimidine 10 is formed from histidine 48 and PLP 49 (37–39). Thiamin biosynthesis in yeast requires fewer enzymes than the bacterial pathway. The biosynthesis of the thiazole requires only one protein (THI4p) in contrast to the bacterial pathway, which requires six (ThiOFSG, IscS and TenI).

Figure 4.

Thiamin pyrophosphate biosynthesis in S. cerevisiae.

All attempts to reconstitute the THI4p-catalyzed reaction, using a variety of C5 carbohydrates, initially failed. However, a breakthrough was achieved by the detection of three metabolites (56, 63 and 64 in Figure 5) released from the protein by heat denaturation (40, 41).

Figure 5.

Mechanistic proposal for the THI4 mediated formation of ADP-thiazole 64.

The identification of product 64 demonstrated that complete thiazole biosynthesis could be achieved using THI4p expressed in E. coli. In addition, this structure demonstrated that the thiazole was adenylated, suggesting that NAD 45, and not a simple pentose, might be the donor of the C5 carbohydrate. Initial attempts to detect Thi4p-catalyzed modification of NAD failed. However, after the structure of THI4p was determined (PDB code = 3FPZ)(42) it was possible to prepare an active site mutant (C204A) that was free of the tightly bound metabolites 56, 63 and 64 (42). This form of the enzyme catalyzed the conversion of NAD 45 and glycine 3 to 56 via intermediates 51 and 52 and confirmed NAD at the C5 carbohydrate donor. (43)

The discovery that metabolite-free THI4p could be isolated when the E. coli overexpression strain was grown at low iron concentrations provided a source of native enzyme with an unoccupied active site. Treatment of NAD and glycine with this form of the enzyme generated intermediate 56. Addition of Fe(III) to this reaction mixture resulted in the transfer of sulfide from Cys205 of THI4p to generate 63 and 64. MS analysis of the protein in this reaction mixture confirmed Cys205 as the sulfide donor.(44) These observations led to the mechanistic proposal outlined in Figure 5.

In this proposal hydrolysis of the N-glycosyl bond of NAD 45 gives 51. Ring opening, tautomerization and imine formation give 53. Tautomerization, loss of water and a second tautomerization generates compound 56, the most labile of the three intermediates released in the heat denaturation experiment. Tautomerization to 57 followed by sulfide transfer from Cys205 of the THI4 protein gives 60. Cyclization and two dehydrations gives the thiazole tautomer 63, the second of the heat released metabolites. A final tautomerization completes the thiazole formation. Our mechanism suggests that the THI4 protein may be a single turnover enzyme. This was confirmed by demonstrating a 1:1 ratio of THI4p to thiamin produced.

In conclusion, we have explored here the mechanistic biochemistry of thiamin thiazole biosynthesis in B. subtilis as a representative prokaryote and in S. cerevisiae as a representative eukaryote. The biosynthetic routes are quite different between the two systems and the reasons for these differences are not yet known. The mechanism of thiazole biosynthesis in bacteria is at an advanced stage, while our understanding of the mechanism of thiazole biosynthesis in yeast is still growing with many unanswered questions remaining. We have not yet identified most of the residues involved in catalyzing the conversion of 45 to 64. We also do not yet understand the role of iron in the sulfur transfer or the physiological role of inactive THI4p.