Supplementary material
Experimental procedures
Recombinant proteins
Mtb H37Rv sucA, dlaT and lpdC genes were amplified by PCR with Pfu DNA polymerase using
primers containing 5 NdeI and 3 NheI sites from genomic DNA. PCR products were digested with
NdeI and NheI, and ligated with T4 ligase into pET11c vectors digested with the same enzymes. E.coli
BL21 (DE3) cells were transformed with pET11c encoding sucA, dlaT or lpdC and grown in 2 liter
at A600 0.5-0.6. SucA expression was induced at 37 C for 4 hours; DlaT was induced at 30 C for 4
c acid; and Lpd was induced at 14 C overnight. Cells were
harvested by centrifugation at 4 C at 5,500g, resuspended in lysis buffer (1 mM PMSF, 1 mM EDTA,
25 mM KPi, pH 7.0 for SucA and DlaT; 20 mM TEA, pH 7.8, 1mM PMSF for Lpd). Cells were broken
by French press at 20,000 psi. Soluble lysates were obtained by centrifugation at 20,000g and were
used to purify overexpressed proteins. SucA was purified to homogeneity through Q-Sepharose
(equilibration buffer and washing buffer: 25 mM KPi, pH 7.0, 1 mM EDTA; gradient elution: 0 to 1 M
NaCl in the same buffer), phenyl-Sepharose (equilibration buffer and washing buffer: 25 mM KPi, 1 M
ammonium sulfate; gradient elution: 1 to 0 M ammonium sulfate in 25 mM KPi), blue-Sepharose
(equilibration buffer and washing buffer: 25 mM KPi, pH 7.0, 1 mM EDTA; gradient elution: 0 to 1 M
NaCl, 1 mM EDTA and 25 mM KPi) and an additional Q-Sepharose column with the same buffer
condition as described above. DlaT was purified through Q-Sepharose with the same buffer conditions
as described above for SucA, and through avidin-agarose column (equilibration buffer and washing
buffer: 25 mM KPi, pH 7.0, 1 mM EDTA; elution buffer: 5 mM lipoic acid, 1 mM EDTA, 25 mM KPi).
Lpd was purified to homogeneity as described (Argyrou and Blanchard, 2001). Eluted fractions
containing recombinant proteins were monitored by either SDS-PAGE or enzymatic assays. Fractions
containing recombinant proteins were combined and concentrated by Centricon 30 (Amicon). Protein
concentrations were measured by Bradford assay. Purified proteins were stored at -20 C. Mtb H37Rv
aceE and pdhC genes were amplified by PCR with Pfu DNA polymerase using primers containing 5
NdeI and 3 NheI sites from genomic DNA. PCR products were digested with NdeI and NheI and
ligated with T4 ligase into pET28b vector digested with the same set of enzymes. E.coli BL21 (DE3)
cells were transformed with pET28b encoding His6 tagged AceE and PdhC and grown in 1 liter culture
in LB media with 50 g/ml kanamycin. Protein expression was induced with 1 mM IPTG at 25 C for 4
hours.
AceE-overexpressing cells were harvested by centrifugation at 4 C at 5,500g, resuspended in lysis
buffer (1 mM PMSF, 25 mM Tris, pH 7.5, 2 mM TPP, 5 mM MgCl2, 500 mM NaCl, 20 mM imidazole)
and broken by French press. Supernatant was loaded on 5 ml Ni+ column (equilibration buffer and
washing buffer as lysis buffer). Proteins were eluted stepwise with 50, 100, 150, 200, 250 and 300 mM
imidazole in 25 mM Tris, pH 7.5, 2 mM TPP, 5 mM MgCl2, 200 mM NaCl. Fractions containing
recombinant proteins were monitored by SDS-PAGE, concentrated in Centricon 50 and dialyzed against
25 mM KPi buffer, pH 7.0, 2 mM TPP, 5 mM MgCl2 and 10 mM NaCl. Omission of TPP in growth
media and purification buffers resulted in inactive protein. PdhC-overexpressing cells were harvested by
centrifugation, resuspended in lysis buffer (1 mM PMSF, 30 mM imidazole, 25 mM Tris, pH 8.0, 500
mM NaCl) and broken by French press. Soluble lysates were loaded on 5 ml Ni+ column (equilibration
buffer: 30 mM imidazole, 25 mM Tris, pH 8.0; washing buffer: 30 mM imidazole, 300 mM NaCl, 25
mM Tris, pH 8.0; elution buffer: 250 mM imidazole, 300 mM NaCl, 25 mM Tris, pH 8.0). Fractions
containing recombinant proteins were monitored by SDS-PAGE. Mtb H37Rv pdhA gene was amplified
by PCR with Pfu DNA polymerase using primers containing 5 NdeI and 3 EcoRV sites. The pdhB gene
was amplified by PCR with Pfu DNA polymerase using primers containing 5 AflII and 3 EcoRI sites
from the same genomic DNA. PCR products were digested and sequentially inserted into pETduet
vector with T4 ligase. E.coli BL21 (DE3) cells were transformed with pETduet encoding both PdhA and
PdhB and were grown in 1 liter of LB in the presence of 50 g/ml kanamycin. PdhA and PdhB were copurified as a complex on Ni+ column as described above for PdhC.
Fig. S1. PH optimum of the reconstituted PDH. Enzymatic reactions were carried out at pH 4.0, 5.0, 6.0,
7.0, 8.0, 9.0, 10, 11 and 12. The Vmax of the reactions is plotted as a function of pH.
NADH (nmole/min/mg)
500
250
0
4
5
6
7
8
pH
9
10
11
12
Fig. S2. Disposition of aceE, dlaT, lpd, pdhA, pdhB and pdhC genes along the chromosome of Mtb
H37Rv. Base pair numbers at the ends of each line indicate how far apart the authentic components of
PDH (aceE, dlaT and lpd) lie from each other on the Mtb chromosome, in contrast to the operon that
was designated pdh.
aceE
acpM
Rv 2242
aceE
f abD
KasB
KasA
accD6
2520890
2510891
ahpE
Rv 2240c
Rv 2239c
dlaT (sucB)
sucB
pepB
lipB
Rv 2219
lipA
Rv 2216
glnA1
2478795
2488794
ephD
lpd
Rv 0459
Rv 0458
Rv 0461
Rv 0460
Rv 0463
icl
lpd
umaA
f adB2
Rv 0466
550311
560310
RV0464c
Rv 0465c
pdhA, pdhB, pdhC
Rv 2492
Rv 2491
Rv 2494
Rv 2493
2811543
2801544
PE_PGRS43
pdhB
pdhC
pdhA
Fig. S3. No PDH activity was reconstituted with PdhA, PdhB, PdhC and Lpd. Production of PdhA and
PdhB (A), and PdhC (B) in E.coli. (A), Fractions of eluted PdhA and PdhB samples from Ni+ column
were analyzed by 10% SDS-PAGE. (B), Recombinant PdhC (1, 2 and 5 g) analyzed by 10% SDSPAGE. (C), PDH activity was tested with 1 M PdhA and 1 M PdhB, 2 M PdhC and 1 M Lpd
(circles); with 1 µM PdhC and 1 µM Lpd (triangles); or with 1 M AceE, 2 M DlaT and 1 M Lpd
(squares).
A
B
C
kDa
kDa
58
111
90
80
61
61
NADH (M)
49
49
36
38
29
0
1
2
3
4
1
2
5 g
0
0.5
Time (min)
1