Chapter 4: Results and Discussion
Results and discussion
4.1
Isolation of panD gene
The 435 bp panD gene was amplified by PCR using Mtb H37Rv BAC222 as template and the primers described in section 3 .1.4. Results from a typical PCR experiment are shown in F ig.1. The predicted 4 35 bp PCR product is evident in lane 2 as is the absence of any DNA product in lane 3 (no template added), signifying that the 435 bp band in lane 2 represents a genuine PCR product. 4.2
Confirmation of expression of panD gene in M. tuberculosis
In the context of studying the Mtb panD as a viable drug target, it was essential to determine the expression of panD protein in Mycobacterium tuberculosis, so as to discount the possibility of the protein being non-existent
or inactive, keeping in mind that gene redundancy and gene decay occur very frequently in pathogenic mycobacterial species (Cole et. al., 2001 ). Thus, the confirmation of expression of panD in Mtb was carried out by eDNA analysis, lmmunoblotting with anti-panD polyclonal antibody using whole cell lysate and lmmuno-Eiectron microscopy. For eDNA analysis, the total Mtb H37Rv RNA was reverse transcribed using random hexamer primers. The resulting eDNA was subjected to PCR using panD gene specific primers. The 435 bp product obtained was cloned into pGEMT-Easy vector and extensive restriction enzyme digestions confirmed the authencity of the product. This result indicates that the panD gene is transcribed in M. tuberculosis. Fig. 4-2 displays the PCR using the eDNA as template. For confirmation of expression of panD at the translational level, western blot analysis of the whole cell extract of M. tuberculosis was performed using antipanD polyclonal antibody (generated in rabbits) to recombinant 6X-His panD. The sera specifically recognized the -16kDa protein in the Mtb extract. The blot was developed using chemiluminiscent substrate (ECL). The result is displayed in Fig. 4-3. The pattern of expression of panD in Mtb was further elucidated using lmmuno gold-electron microscopy. The panD protein was visualized at the surface as well as within the tubercle bacilli. An interesting feature observed 33
Results and discussion
was the variation in the number of gold grains between virulent (H37Rv and Erdman) strain in comparison to attenuated ( H37Ra) strain. The number of gold grains in H37Rv are (40±2), in Erdman are (30±3) whereas in H37Ra are (8±3). These indicate a relation between expression and pathogenicity, although the data is insufficient to assert this conclusively. The electron micrographs are shown in Fig. 4-4. In summary, the lmmunogold electron microscopy shows that panD protein is expressed in Mtb H37Rv, Erdman as well as in H37Ra indicating a universal role in the metabolism in vivo.
4.3
Expression and purification of the 6X-His fusion protein
4.3.1 Generation of fusion construct The 435 bp PCR product, after gel elution, was initially cloned in the pGEMTEasy vector, yielding the vector panDpGEMT. The DNA fragment containing the panD gene was then excised from panDpGEMT using the Nde I and Xho I restriction enzyme sites incorporated in the primers [Lane 2, Fig. 4-5(b)]. After gel purification, the gene was cloned as Nde I I Xho I insert into pET21c expression vector, such that the gene was in-frame with the nucleotide sequence corresponding to six histidine residues at the C -terminus. Double digestion with Nde I and Xho I, of the plasmid carrying the panD fusion gene is expected to produce two DNA fragments of 435 bp (panD gene) and 5.3 kb (pET21 c vector); this is borne out by the band pattern in lane 3 (Fig. 4-5(b )). The panD gene was fully sequenced and found to be identical in sequence to gene available from the database (www.sanger.ac.uk). The fusion gene is expected to encode a protein of 145 aa corresponding to a molecular weight of -16,000 Da. The complete nucleotide sequence, as well as the protein predicted to be encoded by the fusion gene is depicted in Fig. 4-6.
4.3.2 Sequence comparison to other aspartate decarboxylases Multiple sequence alignment of various aspartate decarboxylases namely E. coli, S. coelicolor, C. glutamicum, M. leprae, M. tuberculosis, R. eutropha, A. aeolicus, N. meningitides, B. subtilis, S. aureus, C. jejuni, H. pylori, W. •succinogenes and M. loti, was carried out using the CLUSTALW program
(www.ebi.ac.uk). This is represented in Fig. 4-7. 34
r\v.;)UU.;) OIIU UI.;)I,U.;).;)IVII
The solved crystal structure of E. coli panD protein postulates that the residues lysine, histidine, arginine, threonine and tyrosine, at positions 9, 11, 54, 57, 58 respectively, play an important role in catalysis by panD. All the abovementioned residues are found to be absolutely conserved amongst the enzyme family (Fig. 4-7). Furthermore, Mtb panD shares only 45 % sequence identity with E. coli panD whereas it shares maximum identity (83 %) with its M. /eprae homologue. Additionally, the Mtb panD has 13 extra amino acid
residues at the C-terminus in com pari son to the E. coli homologue. As the crystal structure of E. coli panD shows that the C-terminus of the protein forms a contact point with the other units of the tetramer, the implications of the extra amino acid residues at the C-terminus on the structure-activity relationship are not clear but it is plausible that the extra length contributes to structural stability of the panD tetramer.
4.3.3 Expression'and purification of Mtb 6X-His panD from E. coli After transformation of BL21 (DE3) with panDpET21c, 12-15 g cells (wet weight) were used as the starting material for the purification of recombinant 6X-His panD fusion protein. Essentially, the purification of 6X-His panD protein involved using Ni-NTA affinity column followed by Gel filtration chromatography to determine the oligomeric state of the protein. Fig. 4-8 displays the SDS-PAGE profile of the Ni-NTA affinity purification. A single major protein band of- 16 kDa was obtained after Ni-NTA purification (Fig. 48, lane 6). The purity of the recombinant protein was judged to be - 90-95 %. It is evident from these data that the intended 6X-His panD protein has been successfully expressed and purified based on the positive signals obtained with Ni-NTA eluate using mAbs specific to the 6X-His tag. Western blot analysis of the Ni-NTA purified material, presented in Fig. 4-8 (B), shows that anti-His mAb recognizes the purified recombinant protein and that it exists essentially as the 16 kDa
1t
subunit. An unrelated mAb failed to recognize the
recombinant 6X-His panD (lane 3), consistent with the expectation. Taken together, the data presented in Fig. 4-8 (A-B) demonstrate that recombinant 6X-His panD has been successfully expressed in BL21 (DE3) and purified in a singl~e
step using Ni-NTA chromatography.
35
Results and discussion
4.3.4 Recombinant 6X-His panD exists as a tetramer in solution The Ni-NTA purified recombinant protein (concentrated to 5 IJg/IJI) was loaded onto Superdex-200 (S-200) column to investigate the oligomeric nature of 6XHis panD. Fig. 4-9 displays the elution profile of a purified preparation of 6XHis panD on superdex-200 column. The recombinant fusion protein eluted as a major peak around 13 mins, [corresponding to BSA (64kDa) as a standard] indicating its tetrameric nature. Furthermore, the results from this experiment did not reveal the presence of any monomeric form of the protein. This result is in accordance with the determined crystal structure of E. coli aspartate decarboxylase that shows .the active enzyme as a tetra mer composed of three
a and
p subunits
and an incompletely processed 1t protein. Further, the gel
filtration profile also indicates that the recombinant Mtb 6X-His panD is in its proper confirmation and that protein processing is not essential for tetramer formation.
4.4
Characterization of recombinant aspartate decarboxylase
4.4.1 Formation of active enzyme by autocatalytic cleavage It has been reported in earlier studies (Ramjee et. al., 1997) that the autocatalytic cleavage of E. coli aspartate decarboxylase is temperature assisted. However, the mechanism of this cleavage has not been elucidated.
4.4.1.1 Optimization of temperature for complete autocatalytic cleavage of recombinant 6X-His panD For temperature optimization of 1t protein cleavage, incubation of purified 6XHis panD was carried out at three different temperatures, namely 37 °C, 50 °C and 70 °C for 48 hours. SDS-PAGE (15 %) analysis (Fig. 4-10 (A)) of the incubated samples demonstrated that the protein undergoes autocatalytic cleavage only at 37°C and not at higher temperatures i.e. 50 °C and 70 °C. This is in contrast to earlier work on E. coli panD (Ramjee et. al., 1997), where the protein exhibited increased protein processing at higher temperatures of 50 °C for 48 hours. However, it is rational to assume, that since the optimal growth of M. tuberculosis in defined media and in cell culture occurs at 37 °C, the autocatalytic cleavage should optimally occur at this temperature.
36
Results and discussion
The processing of the
1t
protein was further confirmed by N terminal
sequencing of the cleaved protein. The sequence obtained (Fig. 4-10 (C)), VTIDADLMD, corresponds to theN terminal of the a subunit of Mtb panD. Further confirmation of the autocatalytic cleavage was carried out by lmmunoblotting with anti-His mAbs, taking advantage of the fact that the histidine tag will also be present at the C -terminus of the a s ubumit. Upon processing of the blot, two bands corresponding to the a and
1t
subunits of
Mtb panD were visualized, thus confirming that indeed the protein has been autocatalytically cleaved. A typical result is depicted in (Fig. 4-10 (B)).
4.4.1.2 Optimization of time for complete autocatalytic cleavage of recombinant 6X-His panD To optimize the time taken for complete autocatalytic cleavage of recombinant 6X-His panD, purified protein, incubated for various time intervals of 6 h, 12 h, 24 h, 48 h, 60 hand 72 hat 37 °C, was analyzed by SDS-PAGE (15 %) gel electrophoresis. A typical result is shown in Fig. 4-11. As evident from the gel, the cleavage is essentially complete in 24 hat 37 °C, in contrast to 48 hat 50 °C for E. coli panD (Ramjee et. al., 1997). The time taken by E. coli panD to undergo cleavage is difficult to reconcile with the generation time of an E. coli cell (around 20 min). However, in case of recombinant 6X-His panD, the time taken for autocatalytic cleavage is in accord with the doubling time of M. tuberculosis, which is around 24 h. Thus, the behavior of recombinant 6X-His
panD reflects the natural physiological conditions under which the enzyme may have to regulate the 13-alanine synthesis in the Mtb cell.
4.5
Demonstration of activity of cleaved 6X-His panD
The various methods available for measuring the kinetics of aspartate decarboxylase include trapping radioactive carbon dioxide released from [114C]
aspartate (Cronan, 1980) and also by measuring the release of carbon
dioxide manometrically in a stopped assay (Abell et. al., 1988). However, the above-mentioned
assays
suffer
from
various
containment of radioactivity to exhibiting
drawbacks
low sensitivity.
including
The
kinetic
parameters of Mtb cleaved 6X-His panD were delineated by a new highly
37
Results and discussion
sensitive fluorescence-based assay. In this method, the reaction aliquots, after quenching were derivatized with flourescamine [4-phenylspiro [furan-2 (3H), 1'-phthalan]-3,3'dione], which reacts directly with the primary amines to form flourophores (390 nm excitation, 475 nm emmision) The derivatized mixture
is
then
resolved
on
a
reversed-phase
column
where
the
flourescamine-modified aspartate and f3-alanine are easily resolved (Fig. 4-12 (A)). The main advantage of the assay is its sensitivity coupled with its reproducibility. Using this fluorescence-based assay, the steady state kinetic parameters, Km and
kcat. for recombinant Mtb 6X-His panD were determined to be 219.6 IJM
[(Fig. 4-12, (C)] and 0.65 s· 1 (Fig. 4-12, (D)), respectively. The specific activity of Mtb panD was found to be 2100 nmol/min/mg. These values are in good agreement with those obtained for E. coli panD (Ramjee et. al., 1997). The graphs are depicted in Fig. 4-12.
38
Illustrations
Kb
Fig 4-1.
1
2
3
PCR amplification of panD gene from Mtb H37Rv BAC222 Mtb H37Rv BAC 222 was subjected to PCR using panD gene specific primers. The resultant PCR product was analyzed on a 1.5 % agarose gel. Lane 2 shows the 435 bp PCR product. Lane 3 is the negative control where no template DNA was added . DNA size markers are shown in Lane 1 with the relevant sizes (in Kb) indicated on the left. The arrow on right indicates the position of the panD gene .
1
2
3
5.0-12.2
2.0 1.6
1.0
Fig. 4-2.
PCR analysis of eDNA using panD gene specific primers. eDNA was subjected to PCR with panD specific primers and the resultant products were analyzed on 1.5 % agarose gel. Lane 1 depicts the DNA size markers with relevant sizes (in Kb) indicated on the left. Lane 2 shows PCR product obtained using panD gene specific primers . Lane 3 depicts the product obtained using BAC Rv 222 as positive control. The arrow on the right indicates the positions of the PCR products.
kDa
A
B
94.0. 48.6. Fig 4-3.
36.4•
Western blot of of Mtb whole cell lysate
probed
with
anti-panD
antibodies.
30.0.
Arrow on the right of the panel indicates the position of the panD protein (Lane B) in relation to E. coli expressed Mtb panD
20.6.
(Lane A). The marker sizes (in kDa) are indicated on the left of the blot.
6.60.
A
Fig 4-4.
B
c
lmmuno electron micrographs of H37Rv, Erdman and H37Ra indicating expression of panD. Pre-made ultrathin sections of Mtb H37Rv (A), Erdman (B) and H37Ra (C) were incubated with primary anti-panD antibody followed by gold-labelled anti-rabbit lgG . Samples were contrasted with uranyl acetate and visualized by electron microscopy.
Ndel
(A)
F1 ori panDpet 5874 bp
bla Pst I
Sap/
(B)
Kb
1
2
3
4
5
6
2.0 1.6
1.0
0.5 0.39
0.34
0.29
Fig 4-5. Plasmid vector for expression of panD gene (A)
Vector map of pET21c(+) expression plasmid carrying the panD gene. The panD gene (435 bp) was double digested with Nde I andXho I and inserted into pET21c(+}, downstream of the phage T7 promoter. Ampicillin gene allows the selection in E. coli.
(B)
Miniprep DNA from the putative clones was digested with Nde I and Xho I and run on a 1.5 % agarose gel. Lane 1 and Lane 6 depict the 1 kb DNA marker ladder with relevant sizes (in kb) indicated to the left of the panel. Lane 2 depicts the panDpGEMT whereas Lane 3 is depicting panDpET21c. The double digested panD gene and pET21c(+) vector were run in parallel lanes (lane 4 & 5)
Ndei CATATGTTA CGGACGATGC TGAAGTCGAA GATCCACCGC GCCACGGTGA ML RTML KSK I H R A T V T
CCTGCGCCGA CCTGCACTAC GTCGGCTCGG TGACCATCGA TGCCGACTTG C A D L H Y V G S V T I D A D L ATGGACGCCG CCGACCTGCT GGAAGGCGAA CAGGTAACCA TCGTCGATAT M D A A D L L E G E Q V T I V D I CGACAACGGT GCTCGACTGG TCACCTACGC GATCACCGGC GAACGCGGCA DNG ARLV TYA I T G E R G S GTGGTGTGAT TGGCATCAAC GGTGCCGCCG CGCACTTGGT GCATCCGGGG G V I GIN GAAA HLV HPG GATCTGGTGA TTCTGATTGC GTACGCGACG ATGGACGACG CCCGGGCCCG D L V I LIA YAT MDDA RAR CACATACCAG CCGCGGATCG TGTTTGTCGA CGCTTACAAC AAACCGATCG T Y Q P R I V F V D A Y N K P I D ACATGGGCCA CGATCCGGCA TTTGTGCCCG AAAACGCGGG CGAGCTGCTA M G H D P A F V P E N A G E L L Xhoi GACCCCCGGC TCGGTGTGGG ACTCGAGCAC CACCACCACC ACCACTGA D P R L G V G L E H H H H H H *
Fig 4-6.
Complete nucleotide sequence of the panD gene and the amino acid sequence of the encoded protein
The C-tenninal 6X-His tag is depicted in green
\
I I I
I
S.coelicolor C.glutamicum M.leprae M.tuberculosis E.coli R.eutropha A.aeolicus N.meningitides B.subtilis S.aureus C.jejuni H.pylori W.succinogenes M.loti
MLRTLIKS KI HRATVTQADLHYVGSVTIDADLLDAADLLPGELVHIVDVTNGARLETYV I MLRTILGS KI HRATVTQADLDYVGSVTIDADLVHAAGLIEGEKVAIVDITNGARLETYVI MLRTMLKS KI HRATVTQAYLHYVGSVTIDADLMGAADLLEGEQVTIVDINNGARLVTYAI MLRTMLKS KI HRATVTCADLHYVGSVTIDADLMDAADLLEGEQVTIVDIDNGARLVTYAI MIRTMLQGKLHRVKVTHADLHYEGSCAIDQDFLDAAGILENEAIDIWNVTNGK RFSTYAI MQRIMLRAKLHRVTVTQADLNYEGSCGIDQDLLDAADMKEFEKIELYNVNNGE RFS TY II MLREMLKS KI HRLTVTDADLHYEGSLSLDEYLMELADLKPFEKIDVYNINNGARFQTYVI MFRTMLGGKI HRATVTEADLNYVGS ITVDQDLLDAAGIYPNEKVAIVNNNNGERFETYTI MYRTMMSGKLHRATVTEANLNYVGS ITIDEDLIDAVGMLPNEKVQIVNNNNGARLETY II ----MMNAKI HRARVTESNLNYVGS ITIDSDILEAVDILPNEKVAIVNNNNGARFETYV I MNITLLKS KI HRASVTEARLDYI GS ISIDEKLLQASGILEYEKVQVVNVNNGARFETYTI MTFEMLYS KI HRATITDANLNYI GS ITIDEDLAKLAKLREGMKVEIVDVNNGE RFSTYV I MKFDMLWS KI HRATVTDANLNYVGS ITIDEELMEAAELLVGQKVEILDVNNGERFS TYV I -MRKLVAGKLHGIHVTEANLNYHGS ITLDPDHCEAAGILPMEFVEIWNKNSGARIS TYVI :: . * : * : * : * . * ** . * * *· * * *
60 60 60 60 60 60 60 60 60 56 60 60 60 59
S.coelicolor C.glutamicum M.leprae M. tuberculosis E.coli R.eutropha A.aeolicus N.meningitides B.subtilis S.aureus C.jejuni H.pylori W.succinogenes M.loti
EGERGSGVIGINGAAAHLVHPGDLVILISYAQVTDAEARSLRPRVVHVDGDNRIVGLGAD VGDAGTGNICINGAAAHLINPGDLVIIMSYLQATDAEAKAYEPKIVHVDADNRIVALGND AGERGTGVIGINGAAAHLVHPGDLVILISYGTMEDAEAHAYQPRIVFVDADNKPIDLGHD TGERGSGVIGINGAAAHLVHPGDLVILIAYATMDDARARTYQPRIVFVDAYNKPIDMGHD AAERGSRIISVNGAAAHCASVGDIVIIASFVTMPDEEARTWRPNVAYFEGDNEMKRTAKA KGERGSGEISLNGAAARRAHLGDQLIICTYAPMSDEEIAAYKPKVILVNEKNGIKEIKKF PAPRYSGEVKLNGAAARLGHKGDLIIIASYAQYTEEELENYAPKLIFVNEKNQPVEVKES AGKRGSGVICLNGAAARLVQKSDIVIIMSYVQLSEPEIAAHEPKVVLVDGNNKIRDIISY PGKRGSGVICLNGAAARLVQEGDKVIIISYKMMSDQEAASHEPKVAVLNDQNKIEQMLGN AGERGSGKICLNGAASRLVEVGDVVIIMTYAQLNEEEIKNHAPKVAVMNEDNVIIEMIHE ATQE-EGVVCLNGAAARLAEVGDKVIIMSYADFNEEEAKTFKPKVVFVDENNTATKITNY LGKKRG-EICVNGAAARKVAIGDVVIILAYASMNEDEINAHKPSIVLVDEKNEILEKG-RGERGSREICLNGAAARKVAIGDKIIIVAYAQYDRSELSSYKPTVVLVDEKNDIVQIKHE LGERGSRCCILNGAAARTCQPDDPIIVCNSIYLDEAHITSLKPRIVTFDQDNYILDRLSY : ** * * :: * :* : * *
120 1 20 120 1 20 1 20 120 120 120 120 11 6 11 9 117 120 11 9
s.coelicolor C.glutamicum M.leprae M. tuberculosis E . coli R . eutropha A.aeolicus N.meningitides B . subtilis S . aureus C.jejuni
ASEPVPG---SDQERSPQAVSA------ - - - 1 39 LAEALPG---SGLLTS-RSI------ ---- - 1 36 PGSVPLDISVAAELFDPRIGAR--------- 14 2 PAFVPE NAuEL~DFRLG"'G ---------- - - 13 9 IPVQVA- ----------- ------------- 126
I
H.py1ori W.succinogenes M.loti
TEVK- - ------------------------EPPHTVL-----------------------EPARTIL-----------------------KENTIVL-----------------------EKHGAIF---------- -- -- -- --------
12 4 12 7 127 123 126
v------------------------------
121 SVDLDTDGRYSFSILDEANEALAIPALVSGA 15 0
Fig 4-7. Multiple sequence alignment of the selected aspartate decarboxylases. Sequences were aligned using the CLUSTALW program. Deduced amino acid sequences were obtained from E. coli (GI:12512846), S. coelicolor (GI:14970643), C.
glutamicum (GI:10190283), M. leprae (GI:13092575), M. tuberculosis (GI:2113975), R. eutropha (G1:3786394), A. aeolicus (GI:2983134), N. meningitides (GI:7380140), B. subtilis (GI:1146242), S. aureus (GI:13702554), C. jejuni (GI:6967770), H. pylori (GI:2313109), W
succinogenes (GI:2644972),and M. loti (GI:12044308). The
asterisks and the dots denote the conserved and similar residues, respectively. The conserved glycine-serine residues that are at the site for cleavage in the panD protein are indicated in red. The residues that are thought to play an important role during catalysis by panD are shown in blue. The M. tuberculosis panD contains 13 extra residues at the C-terminus as compared with its E. coli (highlighted).
\
counterpart
\ (8)
(A)
kDa 94. 67.
1
-
2
3
4
5
6
7
kDa 92.
-
43.
52.
30.
30.
1
2
20. 20. 14.
14.
Fig 4-8. Purification and lmmunoblot analysis of recombinant 6XHis panD protein expressed in E. coli BL21 (DE3). (A)
Ni-NTA affinity chromatography purification of recombinant 6X-His panD protein from BL21(DE3} lysate. Samples (Lane 2: Crude lysate; Lane 3: Induced; Lane 4: Flow through; Lane 5: Wash; Lane 6: Elute} were analyzed by denaturing Polyacrylamide gel electrophoresis and visualized by Coomassie staining. Low molecular weight marker was loaded in Lane 1 and their molecular weights (in kDa} are indicated in the left panel.
(B)
Western Blot analysis of Ni-NTA purified recombinant 6X-His panD protein. Aliquots of the purified 6X-His-panD were run on SDS-PAGE (15 %}, transferred to nitrocellulose membrane and probed with anti-His mAbs (lane 1) and an unrelated mAbs (lane 2}. The sizes of the protein marker (in kDa) are indicated on the left side of the blot
Time
Fig. 4-9.
____..
Elution profile of recombinant 6X-His panD on S-200 column Recombinant 6X-His panD protein was chromatographed on a Superdex-200 column (10 X 300 mm) at a flow rate of 0.5 mVmin. The elution profile shown was generated by analyzing fractions by absorbance at 280 nm. The arrows, B (BSA, 13.00 min} and 0 (Ovalbumin, 14.2 min} indicate the position of the markers.
\
(A)
(B)
kDa
1
-......
94·
2
3
kDa
4
43.
2
3
113. 92 .
....
67 •
1
52.
........
30 .
30. 20.
20.
~
. . . . 1t
14·
....1t
.... a.
~
.... a.
14•
(C) AAcid # 1 -') '· 3 4
(A)
(min)
C.'l'ime (min)
( ravJ)
A
8.3 5 13.61 5.75 15 . 68 4 . 01 8.30 4. 03 17.15 13 .32 4.07
8.32 13 . 60 5.76 16 .70 4 . 05 8.32 4.05 17.16 13.32 4 . 05
20.11 17.04 13.44 13.88 10.57 10 . 24 9.37 9.66 3.99 6.80
v T
r
n
6 7
A D L
9 10
Fig 4-10.
R . 'rime
ID
5
8
Pmol
AAcid
l-1 D
505-PAGE (15 %) analysis of the autocatalytic cleavage Freshly purified Mtb 6X-His-pan0 (50Jlg) was incubated for 48 h at three different temperatures (Lane 2: 37 °C, Lane 3: 50 °C, Lane 4: 70 °C). The sizes of the protein markers (in kDa), loaded in Lane 1are indicated on the left side of the gel. The arrows on the right of the panel indicate the position of rc and a subunit.
(B)
Western blot analysis of autocatalytically cleaved recombinant 6X-Hispan0 with anti-His mAb (Lane 2: 37 °C/ 24 h, Lane 3: 70 °C/ 24 h). The sizes of the protein markers (in kDa), loaded in Lane 1 are indicated on the left side of
the gel. The arrows on the right of the panel indicate the
position of rc and a subunit. (C)
N terminal sequence of the autocatalytic cleaved recombinant 6XHis-panD
\
kDa
94. 67. 43.
1
--
2
3
4
5
6
7
8
30.
....7t
20.
.... a
14.
Fig 4-11.
SDS-PAGE (15 %) analysis of time course for autocatalytic cleavage. Freshly purified Mtb 6X-His-panD (50
~g)
was incubated at
37 °C for different time periods (Lane 2: 6 h, Lane 3: 12 h, Lane 4: 24 h, Lane 5: 36 h, Lane 6: 48 h, Lane 7: 60 h, Lane 8: 72 h). The sizes of the protein markers (in kDa), loaded in Lane 1 are indicated on the left side of the gel. The arrows on the right of the panel indicate the position of
n and a subunit.
(A)
-80
>E
1 min L-asp
60 40 1 min fJ- ala
20
Time (min))
(B)
0 E c
CD
c c ..!!
Cl
I ~
240 200 160
•
1.000 mM
o
0.500 mM
• a
0.250 mM 0.125 mM
•
0.050 mM
120 80 ...
40 0
0
2
4
6
8
10
Time (min)
-12.
HPLC analyses of the recombinant Mtb 6X-His panD mediated decarboxylation of L-aspartate (A) Function of time. The positions at which the Fluorescamine derivatized L-aspartate and p-alanine elute are indicated.
(B) Rate curves at increasing amounts of L-aspartate.
(C) 25
~
E
0
20 15
E
.e.
s
10
~5 0 0
300
600
900
1200 1500
[L-asp] ( mM)
(D)
-·-E
0.25
c
"""'0 E
0.20
..&.
-c:: c
0.15
"""'
-20
-15
-10
-5
0
5
10
15
20
1/[L -asp] (mM-1)
12.
HPLC analyses of the recombinant Mtb 6X-His-panD mediated decarboxylation of L-aspartate
C)
D)
Michaelis-Menten plot of Mtb panD activity as a function of L-aspartate concentration obtained using 'best fit 'employing the following equation: Y=Vmax(X)/(Km+X}, where Vmax is the maximal velocity at saturation, Km is the substrate concentration required to reach half-maximal velocity (Vma/2),Y represents the rate, and X is the concentration of L-aspartate. Lineweaver-Burk 'double reciprocal 'plot of 1N versus 1/[S], where V represents the rate of formation of 13-alanine and (S] represents the concentration of the substrate, L-aspartate. The plot was obtained by using the equation:1N = KmNmax (1/[S])+1Nmax·The values obtained were as follows: X intercept (-1/Km) = -4 :552;Y intercept (1Nmax> = 0.04067: slope (K~max> = 0.008933
