Mechanistic studies on thiaminase I: Identification of the product of thiamin degradation in the absence of the nucleophilic cosubstrate

Article abstract of DOI:10.1006/bioo.1999.1156

Thiaminase I catalyzes the decomposition of thiamin under basic conditions in the absence of the nucleophilic cosubstrate. The product of this reaction was identified as the adduct resulting from the displacement of the thiazole moiety of thiamin by the r

Full text of DOI:10.1006/bioo.1999.1156

Bioorganic Chemistry 8, 45–48 (2000)
doi:10.1006/bioo.1999.1156, available online at http://www.idealibrary.com on
Mechanistic Studies on Thiaminase I: Identification of the
Product of Thiamin Degradation in the Absence
of the Nucleophilic Cosubstrate
Min Wu, Elizabeth T. Papish, and Tadhg P. Begley1
Department of Chemistry and Chemical Biology, 120 Baker Laboratory,
Cornell University, Ithaca, New York 14853
Received May 25, 1999
Thiaminase I catalyzes the decomposition of thiamin under basic conditions in the absence
of the nucleophilic cosubstrate. The product of this reaction was identified as the adduct result-
ing from the displacement of the thiazole moiety of thiamin by the ring opened form of thiamin.
© 2000 Academic Press
INTRODUCTION
Thiaminase I catalyzes the cleavage of thiamin by a wide variety of nucleophiles
(Fig. 1) (1). The physiological function of this surprising enzymatic activity, which is
widely distributed in nature, is currently unknown (2).
During our attempts to carry out presteady state kinetics studies on thiaminase I, we
observed turnover of the enzyme in the absence of the nucleophilic cosubstrate (3).
Here, we report the structure of the reaction product formed under these conditions.
EXPERIMENTAL PROCEDURES
General methods and materials. Column chromatography was performed with EM
science silica gel 60 (230–400 mesh). Analytical TLC was performed on silica gel 60
F254-precoated glass plates from EM Science and visualized by UV light. IR spectra
were recorded on a Mattson Galaxy series FT-IR instrument. High resolution FAB
mass spectra were run at the University of Illinois on a 70-SE-4F instrument with a
resolving power of 10,000.
Thiaminase I from Bacillus thiaminolyticus was overexpressed in Escherichia coli
and purified as previously described (1a).
Isolation of the thiaminase I catalyzed thiamin degradation product. Thiaminase I (20 µl,
10.8 mg/ml) was added to a stirred solution of thiamin · HCl (20 mg, 0.06 mmol) in
aqueous NaOH (0.1 M, 1 ml). The reaction mixture (initial pH = 9.1) was stirred at room
temperature for 2 h. TLC analysis (EtOAc:MeOH:AcOH:NH4 OH = 50:48.5:0.75:0.75)
demonstrated the clean formation of 4-methyl-5-(2-hydroxyethyl)-thiazole (Rf = 0.8) and
1 To whom correspondence and reprint requests should be addressed. E-mail:tpb2@cornell.edu.
45
0045-2068/00 $35.00
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All rights of reproduction in any form reserved.

46
WU, PAPISH, AND BEGLEY
FIG. 1. The thiaminase I catalyzed degradation of thiamin.
a new compound (Rf = 0.31). In a control experiment run under identical conditions,
except for the absence of thiaminase, neither product was formed. The reaction mix-
ture was deproteinized by ultrafiltration (Centriprep-10 at 2000 RPM for 30 min) and
lyophilized. The resulting solid was purified by flash column chromatography (silica gel,
EtOAc/MeOH/AcOH/NH4OH, 50:48.5:0.75:0.75) and triturated with CH2Cl2 to give
the product as a white solid (2 mg, 17%). HRMS (FAB in magic bullet) m/z [MH]+ cal-
culated for (C18H26N7O2S) = 404.1869, observed = 404.1869. IR (CH2Cl2) 3329, 3198,
2960, 2918, 2846, 1643, 1589, 1559, 1470, 1428, 1375, 1047 cm−1. The NMR parame-
ters are listed in Table 1.
RESULTS AND DISCUSSION
The molecular formula (C18H25N7O2S) of the product of the thiaminase I catalyzed
thiamin degradation, obtained by HRFABMS, suggested that it was composed of thi-
amin (C12H17N4OS), the pyrimidine moiety of thiamin (C6H8N3), and an additional
oxygen atom. The extra oxygen atom and the presence of an amide carbonyl stretch
at 1643cm−1 suggested that the ring opened form of thiamin (5, Fig. 3) functioned as
the nucleophile in this reaction. The observation of correlations between H6 and H9
in the ROSEY spectrum established that the sulfur and not the pyrimidine nitrogen
functioned as the nucleophile and suggested that the structure of the reaction product
was compound 4 (Fig. 2). This was fully consistent with a detailed analysis of the
NMR spectra (1H, 13C, HMQC, HMBC, ROSEY; Table 1). The product consisted of
a mixture of isomeric amides. NOE measurements demonstrated that the trans amide
was the major product. A similar mixture of isomeric amides had been previously
observed for the ring-opened form of thiamin (4).
A mechanistic proposal for the formation of 4, based on the established mechanism
of thiaminase I and on the high tolerance of the enzyme to variations in the structure
of the nucleophile, is outlined in Fig. 3 (1). The facile base-catalyzed ring opening of
the thiazolium moiety of thiamin generates the nucleophile 5 (4). Reaction of this with
the enzyme bound pyrimidine 7, followed by expulsion of the active site nucleophile,
would give the observed reaction product.
Thiaminase I rapidly looses activity when treated with thiamin in the absence of the
nucleophilic cosubstrate (5). Addition of 4 to the reaction mixture does not result in
inhibition and the mechanism of this inhibition reaction is currently unknown. One
possibility, based on the facile addition of nucleophiles to N1 methyl thiamin (6), is
that N1 of thiamin or of 5 reacts with 7 and that the resulting pyrimidinium cation
alkylates the enzyme.

MECHANISTIC STUDIES ON THIAMINASE I
TABLE 1
47
1H and 13C NMR Chemical Shiftsa and HMQC and HMBC and ROSEY Correlationsb
of the Thiamin Degradation Product
Position
1H NMR δ
13C NMR δ
HMQC
C1
HMBC
C2
ROSEY
H5
1
2
3
4
5
2.36 or 2.38(s)
23.8 or 24.0
166.1
161.8
111.5
154.2
7.70(major, s)
7.81(minor, s)
3.51(br s)
C2, C3, C6
H1, H6, H9
H5, H9
6
7
29.3
132.6(major)
132.2(minor)
138.8(major)
136.1(minor)
33.9
C6
8
9
10
11
2.65(t, J = 6.5 Hz)
3.69(t, J = 6.5 Hz)
1.97(major, s)
1.79(minor, s)
7.30(s)
C9
C10
C11
C7, C8, C10
C7, C9
C7, C8
C7, C8
C13
H5, H6, H10
H9, H17
H12, H17
60.0
18.2
12
165.2
40.2
C12
H11
H17(minor)
H17(minor)
13a
13b
14
15
16
4.50(d, J = 15.2 Hz)
4.35(d, J = 15.2 Hz)
109.5
163.2
167.1
151.8
17
7.73(major, s)
8.15(minor, s)
C13, C15
C16
H10, H11, H18
H13a(minor),
H13b(minor)
H17
18
2.36 or 2.38(s)
23.8 or 24.0
C18
a Recorded in D2O at 500 MHz (1H) and 125 MHz (13C), in ppm.
b HMQC optimized for 1JC-H = 140 Hz. ROSEY mixing time tm 700 ms.
FIG. 2. The structure of the thiamin degradation product formed in the absence of the nucleophilic
cosubstrate.

48
WU, PAPISH, AND BEGLEY
FIG. 3. Mechanistic proposal for the formation of the thiamin degradation product (4).
ACKNOWLEDGMENT
This research was supported by a grant from the National Institutes of Health (DK44083).
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