Biosynthesis

DOI: 10.1002/ange.200504614

Biosynthesis of Thiamin Thiazole: Determinationof the Regiochemistry of the S/OAcyl Shift byUsing 1,4-Dideoxy-d-xylulose-5-phosphate**

Abhishek Chatterjee, Xuemei Han, Fred W. McLafferty,and Tadhg P. Begley*

Thiamin pyrophosphate is an essentialcofactor in all living systems, where itplays a key role in the metabolism ofamino acids and carbohydrates.[1,2] Thebiosynthesis of this cofactor is complexand involves separate syntheses of thethiazole and the pyrimidine moieties,which are then linked to form thecofactor.[3–6] The biosynthesis of thia-min thiazole in Bacillus subtilisinvolves five enzymes and is differentfrom any of the known chemical orbiochemical thiazole syntheses.[7–12] Inthis biosynthesis, ThiF and a cysteinedesulfurase catalyze the formation ofthe sulfide carrier protein thiocarbox-ylate (4, ThiS-COS�), glycine oxidase(ThiO) catalyzes the oxidation of gly-cine to the glycine imine 10, andthiazole synthase (ThiG) catalyzes theformation of thiazole from ThiS-COS� ,the glycine imine 10, and 1-deoxy-d-xylulose-5-phosphate (1,DXP; see Scheme 1).

In the current mechanistic proposal for this reaction[7–12]

(Scheme 1), DXP (1) forms an imine with Lys96 of ThiG.Tautomerization to 3 followed by addition of ThiS-COS� (4)to the resulting ketone gives 5. An O/S acyl shift followed byloss of water gives thioketone 7, which then tautomerizes andeliminates ThiS-COO� to give 9. Addition of this thioenol tothe glycine imine 10, formed in a separate step by ThiO-catalyzed oxidation of glycine, followed by transiminationand decarboxylation, completes the formation of the thiazolemoiety.

This mechanism is supported by the trapping of imine 2,[11]

by the observation of enzyme-catalyzed H/D exchange at C3

and carbonyl oxygen exchange at C2 of DXP (1),[11] by thedemonstration that one of the carboxy terminal oxygen atomsof ThiS-COO� (11) is derived from an oxygen atom fromeither C3 or C4 of DXP,[9] and by the trapping of thioenol 9.[9]

Modeling of intermediate 5 into the active site of the ThiG/ThiS-COO� complex revealed that this enzyme has aremarkably simple active site, using only Asp182 and Glu98to catalyze the conversion of 2 into thiazole phosphate 14.[10]

The regiochemistry of the acyl shift (5!6) has not yetbeen experimentally established. While it is clear that the

carboxy-terminal oxygen atom of ThiS-COO� (11) is notderived from the reaction buffer, the DXP used to demon-strate the direct oxygen transfer from the carbohydrate toThiS-COO� was labeled at both C3 and C4, leaving open thepossibility that the O/S acyl shift from 5 could occur to the C3oxygen substituent rather than to the C4 oxygen atom asdepicted in Scheme 1. While acyl transfer to the C3 hydroxygroup seemed less plausible to us, as it requires the formationof a strained four-membered cyclic intermediate, it could notbe excluded on these grounds because several examples ofenzyme-catalyzed reactions are known in which four-mem-bered cyclic structures are formed involving analogoustetrahedral intermediates.[13–18] It was therefore necessary toexperimentally differentiate between the two pathways forthe cleavage of thioester 5.

Herein, we resolve this ambiguity by using 1,4-dideoxy-d-xylulose-5-phosphate (15, DDXP) as an alternative substratefor ThiG. If this analogue is a substrate, it will form the imine16, which will then tautomerize to the ketone 17 (Scheme 2).Addition of ThiS-thiocarboxylate (4) to this ketone will give18. The subsequent reactivity of this intermediate will thenreveal the regiochemistry of the acyl-transfer reaction: If theacyl transfer occurs to the C3 oxygen atom, 18 will convertinto 19, which will then lose ThiS-COO� to give thioketone20. Reduction of this thioketone with sodium borohydridewill result in the stable incorporation of the ThiS-COS� sulfur

Scheme 1. Proposed mechanism for the formation of the thiazole phosphate moiety of thiaminpyrophosphate. Pi=phosphate.

[*] A. Chatterjee, X. Han, Prof. F. W. McLafferty, Prof. T. P. BegleyDepartment of Chemistry and Chemical BiologyCornell UniversityIthaca, NY 14853 (USA)Fax: (+1)607-255-7133E-mail: tpb2@cornell.edu

[**] This research was supported by grants from the National Institutesof Health to T.P.B. (DK44083) and to F.W.M. (GM16609).

Supporting information for this article, including experimentaldetails, is available on the WWW under http://www.angewandte.orgor from the author.

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atom into the thiazole synthase, as we have previouslydemonstrated in our successful trapping of intermediate 9(Scheme 1). In contrast, if the acyl transfer occurs to the C4oxygen atom, the reaction will be blocked at 18. Intermediate18 will be stable to borohydride reduction and will collapse to17 upon protein denaturation. Therefore, if this experiment iscarried out using [35S]ThiS-COS� , we predict the stable,DDXP-mediated incorporation of 35S into ThiG if the acylshift occurs to the C3 oxygen atom and no incorporation of 35Sinto ThiG if the acyl shift occurs to the C4 oxygen center.

To establish the validity of DDXP (15) as a mechanisticprobe of the ThiG-catalyzed reaction, it was first necessary todemonstrate that it binds to the enzyme in a catalyticallycompetent manner. We used a combination of ThiG-cata-lyzed H/D exchange (on C3) and carbonyl oxygen exchange(on C2) on DDXP and the demonstration that ThiG forms anactive-site imine with DDXP catalytically, to determine ifThiG can process this substrate analogue.

DDXP (15) was prepared in five steps from a-hydroxy-g-butyrolactone as reported previously.[19] To test for theformation of imine 16, ThiG was treated with DDXP followedby reduction with sodium borohydride. Analysis of thisreaction mixture by high-resolution ESI-FTMS (electrosprayionization Fourier transform mass spectrometry) demon-strated the complete conversion of the enzyme into a newspecies with an additional mass of 182 Da corresponding toreduced 16 (Figure 1). Extensive fragmentation of this newspecies localized the imine formation on Lys96, the active-sitelysine. We therefore conclude that DDXP binds to thiazolesynthase and forms an active-site imine.

To test for ThiG-catalyzed exchange of the carbonyloxygen atom of DDXP with buffer, carbonyl-labeled[18O]DDXP was treated with the enzyme and the exchangereaction was monitored by mass spectrometry. Incubation of[18O]DXP with thiazole synthase showed the expectedenzyme-catalyzed exchange (Figure 2A). Incubation of anequimolar mixture of [18O]DXP and [18O]DDXP with thia-zole synthase demonstrated almost equal rates of exchangefor the two compounds (Figure 2B and C). Exchange of thecarbonyl oxygen atom of DXP and DDXP was much slowerin the absence of the enzyme (Figure 2D), which demon-strates that thiazole synthase can catalyze the reversibleformation of imine 16 with DDXP and that DDXP competeswith DXP for the ThiG active site.

Incubation of DDXP with ThiG in deuterated buffer for10 h resulted in the partial exchange of the proton at C3.Figure 3A shows the 1H NMR spectrum of DDXP incubatedovernight in reaction buffer, and Figure 3B shows an identicalreaction mixture in the presence of ThiG in which theresonance for the C3 proton decreases in intensity relative tothat of the C5 proton, which does not exchange and cantherefore be used as an internal standard. The rate of thisexchange is considerably slower than the corresponding rateof exchange of the C3 proton of DXP, which is complete inabout 1 h under identical reaction conditions. This slowerexchange rate is likely due to a combination of factors: theintrinsically lower acidity of the C3 proton of imine 16 relativeto imine 2, and non-optimal orientation of the C3 proton withrespect to the active-site base (Asp182) resulting from the lossof the orientating effect of the C4 hydroxy group.

Scheme 2. Strategy for the use of 1,4-dideoxy-d-xylulose-5-phosphate (DDXP, 15)to determine the regiochemistry of the S/O acyl shift involved in thiazoleformation. P=phosphate.

Figure 1. ESI-FTMS analysis of thiazole synthase modification usingDDXP. A) Mass spectrum of unmodified thiazole synthase afterreduction using borohydride. B) Mass spectrum of thiazole synthasetreated with DDXP followed by reduction using borohydride: Theadduct, with an extra mass of 182 Da, has a mass that is consistentwith reduced 16. C) Extensive fragmentation of the ThiG–DDXPadduct localizes this imine formation to K96, the active-site lysine.The asterisks indicate the peptide fragments with the covalentmodification. Nozzle-skimmer dissociation (NSD) yields amino-ter-minal containing b ions and carboxy-terminal containing y ions.

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The experiments described above demonstrate that thissubstrate analogue binds to the active site of ThiG in acatalytically competent manner. DDXP is therefore a validprobe to differentiate the two mechanisms shown inScheme 2.

Incubation of [35S]ThiS-COS� with DXP in the presenceof ThiG demonstrated the stable incorporation of 35S into theThiG (Figure 4A, lane 2). When the same experiment wascarried out using DDXP, only a trace of sulfur transfer toThiG was detected. (Figure 4A, lane 3). This low level ofsulfur transfer did not increase after incubation overnight(Figure 4A, lane 5), suggesting that failure to form 22 iscaused by the absence of the acyl acceptor at C4 and not dueto a retardation of the O/S acyl-shift reaction. The very lowlevel of DDXP-mediated incorporation of 35S into ThiGshown in lane 3 is likely to arise from low levels of hydrolysisof the thioester 18 during treatment with borohydride. We

therefore conclude that the S/O acyl transfer occurs to the C4hydroxy group of DXP during thiazole formation.

The experiments described herein demonstrate thatDDXP is an alternative substrate for thiazole synthase andcan be used as a mechanistic probe to demonstrate that theThiS acyl-shift reaction occurs to the C4 oxygen atom and notto the C3 oxygen center of DXP (5!6 in Scheme 1). Thesestudies also suggest that DDXP may be useful for thestructural characterization of the active site of thiazolesynthase.

Received: December 29, 2005Revised: March 1, 2006Published online: April 20, 2006

.Keywords: acyl shift · biosynthesis · enzyme catalysis ·isotopic labeling · vitamins

[1] R. F. Butterworth, Nutr. Res. Rev. 2003, 16, 277.[2] F. Jordan, Nat. Prod. Rep. 2003, 20, 184.[3] T. P. Begley, D. M. Downs, S. E. Ealick, F. W. McLafferty,

A. P. G. M. Van Loon, S. Taylor, N. Campobasso, H.-J. Chiu, C.Kinsland, J. J. Reddick, J. Xi, Arch. Microbiol. 1999, 171, 293.

[4] I. D. Spenser, R. L. White, Angew. Chem. 1997, 109, 1096;Angew. Chem. Int. Ed. Engl. 1997, 36, 1032.

[5] B. Estramareix, S. David, New J. Chem. 1996, 20, 607.[6] T. P. Begley, Nat. Prod. Rep. 1996, 13, 177.[7] J.-H. Park, P. C. Dorrestein, H. Zhai, C. Kinsland, F. W.

McLafferty, T. P. Begley, Biochemistry 2003, 42, 12430.[8] E. C. Settembre, P. C. Dorrestein, J.-H. Park, A. M. Augustine,

T. P. Begley, S. E. Ealick, Biochemistry 2003, 42, 2971.[9] P. C. Dorrestein, H. Zhai, F. W. McLafferty, T. P. Begley, Chem.

Biol. 2004, 11, 1373.[10] E. C. Settembre, P. C. Dorrestein, H. Zhai, A. Chatterjee, F. W.

McLafferty, T. P. Begley, S. E. Ealick, Biochemistry 2004, 43,11647.

[11] P. C. Dorrestein, H. Zhai, S. V. Taylor, F. W. McLafferty, T. P.Begley, J. Am. Chem. Soc. 2004, 126, 3091.

[12] H. Zhai, P. C. Dorrestein, A. Chatterjee, T. P. Begley, F. W.McLafferty, J. Am. Soc. Mass Spectrom. 2005, 16, 1052.

[13] D. C. Aldridge, D. Giles, W. B. Turner, J. Chem. Soc. PerkinTrans. 1 1971, 23, 3888.

[14] L. Mayol, V. Piccialli, D. Sica, Tetrahedron Lett. 1987, 28, 3601.[15] L. R. Dick, A. A. Cruikshank, L. Grenier, F. D. Melandri, S. L.

Nunes, R. L. Stein, J. Biol. Chem. 1996, 271, 7273.

Figure 2. Time courses from ESI-MS analysis of the thiazole synthasecatalyzed exchange of [18O]DXP and [18O]DDXP: A) the enzyme-cata-lyzed exchange of [18O]DXP, B) the enzyme-catalyzed exchange of[18O]DXP in the presence of [18O]DDXP, C) the enzyme-catalyzedexchange of [18O]DDXP in the presence of [18O]DXP, and D) theexchange of [18O]DDXP in the absence of enzyme. The rates for 18Oexchange in parts (B) and (C) were measured simultaneously, after anequimolar mixture of labeled DXP and DDXP was added to ThiG.

Figure 3. 1H NMR spectroscopic analysis of the H/D exchange at C3 ofDDXP incubated for 10 h in deuterated buffer: A) in the absence ofthiazole synthase; B) in the presence of thiazole synthase. The protonat C3 and those at C5 resonate at d=4.35 and 3.76 ppm, respectively.

Figure 4. Analysis of the transfer of 35S from [35S]ThiS-COS� to thiazolesynthase (ThiG) by SDS-PAGE/autoradiography. A) Lane 1: ThiG +[35S]ThiS-COS� , 30 min; lane 2: ThiG + [35S]ThiS-COS� + DXP,30 min; lane 3: ThiG + [35S]ThiS-COS� + DDXP, 30 min; lane 4:ThiG + [35S]ThiS-COS� , 12 h; lane 5: ThiG + [35S]ThiS-COS� +DDXP, 12 h. B) Magnified image of the region highlighted in part (A).

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[16] B. O. Bachmann, R. Li, C. A. Townsend, Proc. Natl. Acad. Sci.USA 1998, 95, 9082.

[17] M. T. Miller, B. Gerratana, A. Stapon, C. A. Townsend, A. C.Rosenzweig, J. Biol. Chem. 2003, 278, 40996.

[18] B. Gerratana, A. Stapon, C. A. Townsend, Biochemistry 2003, 42,7836.

[19] J.-F. Hoeffler, D. Tritsch, C. Grosdemange-Billiard, M. Rohmer,Eur. J. Biochem. 2002, 269, 4446.

[20] S. V. Taylor, L. D. Vu, T. P. Begley, U. Schoerken, S. Grolle, G. A.Sprenger, S. Bringer-Meyer, H. Sahm, J. Org. Chem. 1998, 63,2375.

[21] S. C. Beu, M. W. Senko, J. P. Quinn, F. M. Wampler III, F. W.McLafferty, J. Am. Soc. Mass Spectrom. 1993, 4, 557.

[22] H. Zhai, X. Han, K. Breuker, F. W. McLafferty, Anal. Chem.2005, 77, 5777.

[23] D. M. Horn, R. A. Zubarev, F. W. McLafferty, J. Am. Soc. MassSpectrom. 2000, 11, 320.

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