Supporting Table 1; Molecular size and characteristics of uridine diphosphate
glucose 4-epimerse purified from various sources
Resource
References
Molecular size of
Cofactor
subunit* (kDa#)
required
Characteristics
Escherichia coli
1
79
none
NAD+ contained
Kluyveromyces
2
125
none
NAD+ contained
3
183
none
NAD+ contained
Wheat germ
4
60
NAD+
--
Bovine
5
60
NAD+
--
Porcine
6
88
NAD+
--
fragilis
Saccharomyces
cerevisiae
* All the enzymes are composed of two identical subunits. # kilodaltons.
1. Wilson DB & Hogness DS (1964) The enzymes of the galactose operon in
Escherichia coli. I. Purification and characterization of uridine
diphohosphogalactose 4-epimerase. J. Biol. Chem. 239: 2469-2481.
2. Darrow RA & Rodstrom R (1968) Purification and characterization of uridine
diphosphate galactose 4-epimerase from yeast. Biochemistry 7: 1645-1654.
3. Fukasawa T, Obonai K, Segawa T & Nogi Y (1980) The enzymes of the galactose
cluster in Saccharomyces cerevisiae. II. Purification and characterization of uridine
diphosphoglucose 4-epimerase. J Biol Chem. 255: 2705-2707.
4. Fan DF & Feingold DS (1969) Nucleoside diphosphate-sugar 4-epimerases I. Uridine
diphosphate 4-epimerase from wheat germ. Plant Physiol. 44: 599-604.
5. Tsai CM, Holmberg N & Ebner KE (1970) Purification, stabilization, and properties
of bovine mammary UDP-galactose 4-epimerase. Arch. Biochem. Piophys. 136:
233-244.
6. Piller F, Hanlon MH & Hill RL (1983) Co-purification and characterization
of UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase from
porcine submaxillary glands. J. Biol. Chem. 258: 10774-10778.
Supporting Figure 1. Construction of Gal4p-dependent expression plasmid for
Gal10p in Kluyveromyces lactis.
Kl-ars stands for automatically replicating sequence of K. lactis and is derived from
plasmid KARS101 (Irene et al., 2004). TRP1 is derived from an S. cerevisiae plasmid
pJJ246 (Jones & Prakash, 1990).
The promoter region including UASG and GAL1p
(upstream activating sequence and the transcription initiation site, respectively) is
derived from p4XX (Mumberg et al., 1994).
ADH1t is derived from pVT102U
(Vernet et al., 1987), which encompasses the transcription termination site.
Shaded
region at the N-terminus of GAL10 represents histidine-tag containing the target peptide
of protease Factor Xa (in red), which comes from the cloning/expression region of
pET16b (Novagen Co.).
The amino acid sequence is shown in the uppermost area.
Supporting Figure 2. Restriction map of Kluyveromyces lactis GAL10 and its
deletions.
The restriction sites of GAL10 are deduced from the published sequence in the database
(http://cbi.labri.fr/Genolevures/). The introduction of deletion covering both epimerase
and mutarotase domains were carried out by three steps: 1) The GAL10 gene,
PCR-cloned from K. lactis KA5-6C (MATa ade his leu) DNA, was inserted to
pBluescript SK(+) between XhoI and HindIII (pBlue-KlGAL10).
2) The
hisG-URA3-hisG region excised from pNK51 (Alani et al., 1987) with BglII and BamHI
was inserted in pBluescript SK(+) at the BamHI site (pBluePO-URA3).
3) The
BglII-SalI region of pBlue-KlGAL10 was replaced with the XhoI and BamHI fragment
from pBluePO-URA3. 4) The XhoI-PvuII fragment of the resultant plasmid,
pBlueKl-gal10::PO-URA3, was used for transformation of JA6 (MATa, ade1-600
trp1-11 ura3-12) to select Ura+ colonies. Successful disruption of GAL10 was
confirmed by galactose-negative phenotype of transformants, from which 5-fluoroorotic
acid-resistant clones were selected to create JA6gal10.
Introduction of deletion to
the mutarotase domain was carried out by removing the PstI-HpaI fragment from
GAL10; precisely, the cloned GAL10 was cleaved first with PstI, end-blunted with T4
DNA polymerase, and with HpaI. The resultant ends in GAL10 were ligated to yield
pK411.
Supporting Figure 3. Linearity of polarometric assay of mutarotase
a) Linearity vs. time
0.300
log(0-e)/(t-e)
0.250
No enzyme
gal80Dgal10D 100 mcL
gal80D 25 mcL
gal80D 50 mcL
gal80D100 mcL
0.200
0.150
0.100
0.050
0.000
0
2
4
6
8
10
Time in minute
b) Linearity vs. amount of enzyme used
0.040
0.035
Rate constant
0.030
0.025
0.020
0.015
0.010
0.005
0.000
0
20
40
60
80
100
Amount of enzyme in microliter
a) The enzyme sample used was the same crude extracts described in Table 1 in the text,
which was prepared from Kluyveromyces lactis strain JA6gal80gal80D) and
JA6gal80gal10 (gal80Dgal10D) grown on glucose. b) The rate constant was
calculated from the slope of log (0-e)/(t-e) for the crude extract of JA6gal80 in
a), where 0, e and t are rotation at 0-time, equilibrium and time t, respectively. mcL
stands for microliter.
1
y = 0,2326x + 0,1908
2
R = 0,9841
1/V (min mg dry weight nmol-1)
0,8
0,6
HGT1
GAL2
0,4
0,2
y = 0,2331x + 0,1622
2
R = 0,982
0
-2
-1
0
1
2
3
4
-0,2
1/S (mM [1-14C]D-galactose-1)
Supporting Figure 4. Lineweaver-Burk plot showing galactose uptake in the
strain 2359lac12hgt1 (Baruffini et al., 2006) transformed with either
KCp491-HGT1 (black line and squares) or KCp491-GAL2 (grey line and
squares). Galactose uptake was measured as previously reported (Baruffini
et al., 2006), except that uptake activity was measured after 10 s of
incubation with [1-14C]-D-galactose at final concentrations ranging from 0.5
to 10 mM. For each concentration, the value comes from the subtraction of
the uptake activity of the involved strain with the background uptake
activity of strain 2359lac12hgt1 transformed with KCp491. Values are
means of three independent uptake experiments.
Supporting Figure 5. GAL10-independent mutarotase activity in concentrated
crude extracts and ammonium sulfate precipitates from JAgal10gal80
Absorbance at 340nm
0.5
0.4
0.3
Crude extract
55% ppt
0.2
80% ppt
0.1
0.0
0.0
2.0
4.0
6.0
8.0
10.0
Time in minute
The assay was carried out according to Bouffard et al. (1994). Reaction was started by
the addition of -glucose solution, which is prepared immediately before each assay.
The absorbance of the no enzyme control was subtracted automatically by setting a
reference cuvette without sample in a dual beam spectrophotometer (Hitchi). The
protein concentration of crude extracts, ammonium sulfate precipitates at 0% to 55%
(55%ppt), and 55% to 80% (80% ppt) was 7.1 mg/ml, 4.7 mg/ml, and 2.5 mg/ml,
respectively. The amount of enzyme used is 10 l each of crude extract, 10-times
diluted 55% ppt, and 10-times diluted 80% ppt.
References for Supporting Information
Baruffini E, Goffrini P, Donnini C & Lodi T (2006) Galactose transport in
Kluyveromyces lactis: major role of the glucose permease Hgt1. FEMS Yeast Res.
6: 1235-1242.
Bouffard GG, Rudd, KE & Adhya, SL (1994) Dependence of lactose metabolism upon
mutarotase encoded in the gal operon in Escherichia coli. J. Mol. Biol. 244:
269-278.
Irene C, Maciariello C, Cioci F, Camilloni G, Newlon CS & Fabiani L (2004)
Identification of the sequences required for chromosomal replicator function in
Kluyveromyces lactis. Molec Microbiolog 51: 1413–1423.
Jones JS & Prakash L (1990) Yeast Saccharomyces cerevisiae selectable markers in
pUC18 polylinkers. Yeast 6: 363-366.
Mumberg D, Mueller R, & Fink M (1994) Regulatable promoters of Saccharomyces
cerevisiae: comparison of transcriptional activity and their use for heterologous
expression. Nucleic Acids Research: 22, 5767-5768.
Vernet T, Dignard D & Thomas DY (1987) A family of yeast expression vectors
containing the phage f1 intergenic region. Gene 52: 225-233.