Reactivity

In addition to their highly similar activation mechanism, the donor reactions resulting in formation of the Breslow intermediate are alike along the family of ThDP-dependent enzymes (Fig. 3). Upon encounter, a carbonyl-harboring substrate is nucleophilically attacked by the activated C2. By internal acid-base catalysis, most probably by the N4’, the carbonyl oxygen is reduced and a covalent, tetrahedral intermediate is formed.

Subsequently, one substituent of the C2α is removed, either in a decarboxylation reaction or once again by acid-base catalysis, forming the Breslow intermediate.

From there on, the pathways diverge depending on the second substrate. There are

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CH₃: Acetylphosphate (POX, XFPK) CH₃: Acetyl-dihydrolipoic acid (PDH) CH₃: Acetyl-CoA (PFOR)

CH₃: Acetyaldehyde (PDC) Phe: Benzaldehyde (BAL; BFDC)

(CH2)5CHO: 6-oxohexaonoate (CDH)

CH₂OH: C5-C7 ketose sugars (TK) CH₃ : 2-acetolactate (AHAS)

Figure 3. Shared reaction steps of ThDP-dependent enzymes and pathway diver-gence. The C2-H is abstracted by a base, forming the activated ylidene species. This species attacks an electrophilic carbon atom, and by base-assisted rearrangement and bond cleavage forms the Breslow intermediate. R₁ denounces the aminopyrimidine moiety in ThDP, R₂ the pyrophosphate anchor in ThDP, R₃ and R₄ denote the sub-stituents neighboring the carbonyl function. The multiple pathways diverging from the Breslow intermediate are shown below, indicating common substrates and the different products according to the identity of R₃, as well as the catalyzing enzymes. The C2αis denoted in red.

at least 18 different ThDP-dependent enzymes described, belonging to the enzyme classes of oxidases, transferases, hydrolases and lyases.

Oxidases catalyze the oxidation of their substrate using electron acceptors such as phos-phate (e.g. pyruvate oxidase (POX)), lipoamide (e.g. pyruvate dehydrogenase (PDH))

or coenzyme A (e.g. pyruvate:ferredoxin oxidoreductase (PFOR)). They often employ additional cofactors such as flavin adenine dinucleotide (FAD) or iron-sulfur clusters.

The transferases commonly employ aldehyde-containing acceptor substrates, linking the C2α of the Breslow intermediate to the acceptors carbonyl atom (e.g. transketolase (TK), acetohydroxyacid synthase (AHAS)). However, there are exceptions, for example acetoin dehydrogenase, which uses CoA as an acceptor substrate and reduces NAD⁺ to NADH. Cyclohexane-1,2-Hydrolase is the only known ThDP-dependent hydrolase, in so far unique as it does not cleave off any part of the product after formation of the covalent adduct, by virtue of its circularity (Steinbach et al., 2011; Steinbachet al., 2012). The final group are the lyases. They cleave their respective substrates, often decarboxylating it (e.g. pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL)).

However, some of them still use acceptor substrates, such as phosphate (e.g. xylu-lose/fructose phosphoketolase (XFPK)). They, as well as the transferases, are of special biotechnological interest as they frequently display a relatively broad substrate spectrum and can be modified to accept uncommon compounds (Mülleret al., 2013).

One common question is how this broad reaction and substrate specificity is modulated.

The most obvious answer is by variations in the active site, which enable access for substrates of varying sizes and properties, and modify the stereo- and enantioselectivi-ties (Haileset al., 2013; Westphalet al., 2014; Wechsleret al., 2015; Affaticatiet al., 2016).

Recent research revealed an additional possibility to control the reaction pathway, which was disregarded for a long time. As shown in figure 3, the shared intermediate in ThDP-dependent enzymes is assumed to exist as the enamine and carbanion tautomers, while formation of a keto-form was assumed to cause breaking of aromaticity of the thiazolium ring. As such, it was regarded as thermodynamically unfavorable (Breslow, 1958). However, in the last decade evidence accumulated supporting the existence of this intermediate. In solution studies of NHCs revealed a ketone species to be the thermodynamic minimum of the reaction of 1,3,4-triphenyl-4,5- dihydro-1H-1,2,4-triazol-5-ylidene with propionic aldehyde in tetrahydrofuran (Fig. 4A)(Berkesselet al.,

S

Figure 4. Putative ketone-intermediates in NHCs and ThDP-dependent enzymes.

(A)The intermediate generated using 1,3,4-triphenyl-4,5- dihydro-1H-1,2,4-triazol-5-ylidene and propionic acid in THF (Berkessel et al., 2010). (B)The putative radical species derived from the intermediate observed in crystalloofLpPOX (Meyeret al., 2012). (C)Theσ/n cationic radical ofDafPFOR, observedin crystallo (Amaraet al., 2007).

2010). Additionally, and in the context of biological systems much more substantial, a similar intermediate was observed in two different ThDP-dependent enzymes. In pyruvate:ferredoxin oxidoreductase (PFOR), a stableσ/n cationic radical was captured in crystallo. The structure showed significant distortion of the thiazolium ring, indicating loss of aromaticity, as well as generation of a keto-function at the C2α, albeit the character of the radicalin vivois subject to discussion, as spectroscopic studies point towards a π-type radical (Fig. 4C)(Chabriere, 2001; Cavazzaet al., 2006; Amaraet al., 2007).

In pyruvate oxidase, a tetrahedral, covalent hydroxyethyl-ThDP intermediate was ob-served, where the C2α-Oαbond showed at least partial double bond character. While this specific intermediate would be incompetent in the native reaction, it suggests the presence of an acetyl radical species, where the radical is localized at the C2, opposed to the precedent model employing a hydroxyethyl-radical, with radical-localization at the C2α. It is suggested that the second substrate phosphate nucleophilically attacks the radical intermediate at the C2α. In the old model it would attack an electron rich center, whereas in the new model it would attack a carbonyl carbon (Fig. 4B)(Meyeret al., 2012). All these results suggest that ThDP-dependent enzymes employing nucleophilic or electron rich substrates are capable of forming intermediates with an electrophilic C2α. As the formation of this intermediate would be detrimental in enzymes catalyzing carboligations, ThDP-dependent enzymes seem to have the capability to stabilize the

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Figure 5. Synthesis of ThDP and MeOThDP in E. coli. Hydroxymethylpyrimidin (HMP, R: Me) or bacimethrin (R: OMe) are transformed into ThDP (R: Me) or MeOThDP (R: OMe) by the native E. coli thiamine synthesis machinery. The HMP-P kinase ThiD transforms the pyrimidine-moiety into the respective pyrophosphate derivative, the thiamine phosphate synthase ThiE links it to the 4-methyl,5-hydroxyethyl thiazole phosphate moiety and the thiamine phosphate kinase ThiL phosphorylates the joint molecule to yield ThDP (adapted from Reddicket al., 2001)

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respective intermediate by virtue of their active site.

As stated before, until today, no derivatives of ThDP with a similar effectivity were discovered, as all investigated substitutions at different positions of ThDP resulted in a severe loss of activity in the respective model enzymes (Schellenberger and Winter, 1960; Schellenberger et al., 1966a; Schellenberger et al., 1966b; Schellenberger, 1967; Schellenberger et al., 1967). This immutable character of ThDP is exploited by the bacterial species Bacillus megaterium, Streptomyces albus and Clostridium botulinum (Tanaka et al., 1962; Drautz et al., 1987; Cooper et al., 2014). These produce a compound called bacimethrin (Fig. 5), which was shown to be an inhibitor for bacterial growth. It was shown that the toxicity does not stem from an inhibition of the ThDP biosynthesis pathway, as bacimethrin is incorporated at an up to 6-fold elevated rate compared to the natural precursor hydroxymethylpyrimidin, forming 2’-methoxythiamine diphosphate. In the same study, E. coli transketolase, deoxy-D-xylulose-5-phosphate synthase (DXPS) andα-ketoglutarate dehydrogenase (KGDH) were identified as major inhibition targets in vivo by the produced methoxythiamine (Reddicket al., 2001). This was later supported byin vitrostudies for DXPS, while the ThDP containing E1 component of the KGDH-complex proved to be resistant against MeOThDP, opposed to E1 of the pyruvate dehydrogenase complex, which showed a loss of activity of around 90 % (Nemeriaet al., 2016). As of now, the mode of action by which this reduction in activity is achieved is unknown.