rSHMT_EJB_1997

Eur. J. Biochem. 247, 372-379 (1997)
0FEBS 1997
Importance of the amino terminus in maintenance of oligomeric structure
of sheep liver cytosolic serine hydroxymethyltransferase
Junutula Reddy JAGATH', Balasubramanya SHARMA', BrahatheeswaranBHASKAR', Asis DATTA', Naropantul Appaji RAO'
and Handanahal S. SAVITHRI'
' Department of Biochemistry,Indian Institute of Science, Bangalore, India
* School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
(Received 19 Febmary/24 April 1997) - EJB 97 0266/4
The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase
(SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14,
30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion
mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-
based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT
and des-(A1-W14)-SHMT were present in the soluble fraction and they were purified to homogeneity.
The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low
levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-
(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-
SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-
independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate.
The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However,
at 55"C, the des-(Al-W14)-SHMT lost almost all the activity within 5 min, while at the same temper-
ature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denatur-
ation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52"C) com-
pared to rSHMT (56°C) and des-(A1-K6)-SHMT (55"C), suggesting that N-terminal deletion had re-
sulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the
enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2-CO.l M) in the case
of des-(Al-W14)-SHMT compared to rSHMT (1.8?0.1 M) and des-(Al-K6)-SHMT (1.7-C0.1 M).
The apoenzyme of des-(Al-W14)-SHMT was present predominantly in the dimer form, whereas the
apoenzymes of rSHMT and des-(Al-K6)-SHMT were a mixture of tetramers ( ~ 7 5 %and =65%, re-
spectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted
upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-
W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained corre-
lated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetra-
meric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition
to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.
Keywords: sheep cytosolic serine hydroxymethyltransferase ; N-terminal deletion mutant; C-terminal de-
letion mutant; overexpression; subunit assembly.
Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-
phosphate (pyridoxal-P)-dependent enzyme, plays a crucial role
in one-carbon metabolism and provides precursors for the bio-
synthesis of purines, thymidylate, methionine, etc. [11. The
physiological reaction catalyzed by this enzyme is the reversible
conversion of serine and 5,6,7&tetrahydrofolate (H,-folate) to
Correspondence to H. S. Savithri, Department of Biochemistry,
URL: http ://biochem.iisc.ernet.in
Abbreviations. SHMT, serine hydroxymethyltransferase; H,-folate,
5,6,7,8-tetrahydrofolate;pyridoxal-P, pyridoxal 5'-phosphate; pyridox-
mine?,pyridoxamine 5'-phosphate; rSHMT, sheep liver cytosolic re-
combinant SHMT; AAT, aspartate aminotransferase; buffer A, 50 mM
potassium phosphate, pH 7.4, 1 mM 2-mercaptoethanol, 1 mM EDTA;
buffer B, buffer A with SO pM pyridoxal-P.
Enzymes. Serine hydroxymethyltransferase (EC 2.1.2.1); aspartate
aminotransferase(EC 2.6.1.1).
Indian Institute of Science, Bangalore-560012,India
glycine and 5,10-methylene-H4-folate. The enzyme exhibits
broad substrate and reaction specificity [2]. The mammalian cy-
tosolic enzyme is a tetramer of identical subunits of 53 kDa with
4 mol pyridoxal-P/mol enzyme [3]. The sheep liver cytosolic
enzyme has been extensively characterized in our laboratory
with respect to its primary structure [4], catalysis [5,61, thermal
stability [7] and interaction with inhibitors [8- 101. This enzyme
has been cloned and overexpressed in Escherichia coli [ll]. Re-
cently, it has been shown that pyridoxal-P has an important role
in the stabilization of quaternary structure [12].
In pyridoxal-P-dependent enzymes, limited proteolysis re-
sults in the formation of a core protein devoid of N-terminus
which is either partially active [13], inactive [I41 or fully active
[15, 161. In the case of porcine cytosolic aspartate aminotrans-
ferase (AAT), the deletion of nine amino acids from the amino-
terminus by oligonucleotide-directed mutagenesis resulted in a
striking decrease in its activity and thermal stability [17]. The

Reddy Jagath et al. ( E m J. Biochem. 247) 373
N-terminal region was shown to play a role in the inter-subunit
interactions of porcine cytosolic AAT [17]. It was evident from
the crystallographic data that several pyridoxal-P-requiring en-
zymes have the same spatial fold as AAT even though these
proteins have less than 15% sequence identity [18-201.
The alignment of SHMT sequences from several sources re-
vealed that the N- and C-termini were less conserved compared
to rest of the protein [4]. It was therefore of interest to examine
the role of these terminal regions of the protein on its structural
and functional properties. By limited proteolytic digestion, it
was shown that the N-terminal 15residues may not be important
for the catalytic activity ([16] and Bhaskar et al., unpublished
results). A disadvantage of the partial proteolysis procedure is
the difficulty in obtaining pure preparations of the truncated pro-
tein as the cleaved peptides often associate non-covalently with
the core molecule. To circumvent this problem, we decided to
generate the deletions at 5’ and 3‘ ends of the cDNA clone of
SHMT and express these deletion clones. In this paper, we report
the construction, expression, purification, characterization of de-
letion mutants of the sheep liver cytosolic SHMT and also show
the importance of N-terminal arm in the stabilization of the di-
mer-dimer interactions which are crucial for tetramer formation
and cofactor binding.
EXPERIMENTAL PROCEDURES
Materials. [a-3ZP]dATP(3000 Ci/mmol) and ~-[3-’~C]serine
(55 mCi/mmol) were obtained from Amersham International.
Restriction endonucleases and DNA-modifying enzymes were
purchased from New England Biolabs and Amersham Interna-
tional. Blue-Sepharose, CM-Sephadex and Sephacryl S-200
were obtained from Pharmacia Fine Chemicals. All other bio-
chemicals used in this study were obtained from Sigma Chemi-
cal Company. H,-folate was prepared by the method of Hatefi
et al. [21]. The oligonucleotides were synthesized by Bangalore
Genei Private Ltd., (Bangalore, India). Centricon filters were
from Amicon Inc.
Bacterial strains and growth conditions. E. coli strain
DH5a (BRL) was the recipient for all the plasmids used in sub-
cloning. The BL21(DE3), BL21(DE3) pLysS strains [22] were
used for the bacterial expression of pETSH and deletion clones.
Luria-Bertani medium or terrific broth with 50 pg/ml of ampicil-
lin was used for growing E. cnli cells containing the plasmids
WI.
DNA manipulations. Plasmids were prepared by the alka-
line lysis procedure as described by Sambrook et al. [23]. Re-
striction endonuclease digestions, Klenow filling and ligations
were performed according to the manufacturer’s instructions.
The preparation of competent cells and transformation were per-
formed by the method of Alexander et al. [24]. From the agarose
gel, the DNA fragments (>200 bp) were eluted by Gene Clean
I1 Kit (Bio 101), <200 bp in size by Mermaid Kit (Bio 101) or
by low-melting agarose gel method [23].
Construction of N and C-terminaldeletion clones. A com-
mon vector for the expression of mutant clones was prepared by
the following method. PET-3c vector [25] has two NheI sites.
To make a unique NheI site which is next to the ATG, the second
NheI site was removed by deleting a 200-bp fragment by EcoRV
digestion and self ligation. This vector was designated as PET-
3e. The PET-3e plasmid was digested with NheI, Klenow-filled,
then cut with BarnHI and gel purified as described in the previ-
ous section.
pETSH expression construct 1111 was used for all the DNA
manipulations. This pETSH clone was cleaved by NdeI, KpnI
and BamHI into two fragments i.e. a 1.26-kb KpnI-BamHI as
constant fragment and a 230-bp of NdeI-KpnI as variable frag-
ment. For convenient handling, the NdeI- KpnI insert size was
increased by releasing the insert either with SphI- EcoRI
(560 bp) or BglII-EcoRI (350 bp). The SphI-EcoRI and
BglII-EcoRI fragments were gel purified and used for further
modifications. The SphI-EcoRI fragment has an unique AvaII
site at position 45 bp from the ATG codon. To obtain a 14-
amino-acid deletion clone, the SphI- EcoRl fragment was di-
gested with AvaII, Klenow filled followed by KpnI digestion.
This AvaII-KpnI 180-bp fragment along with KpnI-BamHI
1.26-kb constant fragment was ligated to PET-3e vector at
BamHI and end-filled NheI sites. This construct was designated
as pSN2 (414). Similarly, a BglII-EcoRI 370-bp fragment was
used in the construction of pSN3 (430) and pSN4 (449) deletion
clones. In order to obtain a large N-terminal deletion (473,
pETSH was digested with Acc651, Klenow filled, followed by
the BarnHI digestion. This 1.26-kb end-filled Acc65I-BamHI
fragment was cloned into the BarnHI and end-filled NheI sites
of PET-3e vector (designated as pSN6). pSC2 (4185) C-terminal
deletion clone was constructed by cloning the BglII-StuI 1.0-
kb fragment at the BglII and end-filled XbaI sites of pETSH
vector. Two oligonucleotides were designed, namely SHPla (5’
TG GCA CCC AGA GAT GCC GAT 3’) primer for a six amino-
acid deletion (excluding Metl) and SHPlb (5’ TG GCT GTT
CTG GAG GCC CTA 3’) primer for a 58-amino-acid deletion
(excluding Metl). PCR was performed using Vent DNA poly-
merase as described in New England Biolabs product specifica-
tions with the following modifications. For a 100-p1 reaction,
10 ng pETSH plasmid, 0.15 pM of each primer (SHPla and
SHMl [Ill) and 0.4 mM of each dNTP were used. The sample
was incubated in a thermal cycler (MJ Research) at 94°C for
4 min then kept for 25 cycles of amplification. Each cycle in-
cluded I-min denaturation (94”C), I-min annealing (50°C) and
1.5-min chain elongation (72°C). After 25 cycles, the reaction
was extended for an additional 5 min at 72°C. The 1.43-kb PCR
product was gel purified from the agarose gel. This product was
digested with KpnI and the 210-bp fragment was gel purified
and cloned at KpnI and end-filled NdeI sites of pETSH vector,
designated as pSNl (46). Similarly the PCR was performed
using SHPlb and SHMl primers and the 1.30-kb PCR product
was gel purifiei. This was digested with KpnI and the 52-bp
fragment was cloned at the KpnI and end-filled NdeI sites of
pETSH vector, designated as pSN5 (458). The clone pSCl
(449) was constructed by using SHPla and SHM2 (5’ TACAG,
GATCCTAGATCTGCAGGGTCA 3’) with a stop codon at
435th amino acid followed by a BamHI site, as primers. The
cloning junctions and the presence of deletion for all the above
deletion clones were confirmed by restriction enzyme digestions
and DNA sequencing [26].
Expression of N- and C-terminal deletion clones in BL21
(DE3) pLysS. BL21(DE3) pLysS strain with the deletion con-
structs, pETSH, PET-3e plasmids were grown in 25 ml Luria-
Bertani medium containing 50 lg/ml of ampicillin and induced
by addition of (0.3 mM) isopropyl 1-thio-P-galactopyranoside.
The total, soluble and insoluble extracts were analyzed on SDS/
PAGE [27].
Protein sequencing. The expressed proteins were analyzed
on SDS/PAGE and transferred to a Hybond Poly(viny1idene
difluoride) membrane. The proteins were stained with Ponceau
S and the appropriate bands were cut out, loaded on to a Shi-
madzu gas-phase sequenator PSQl to determine the N-terminal
sequence of the mutant proteins [28].
Purification of sheep liver cytosolic recombinant SHMT
(rSHMT), des-(Al-K6)-SHMT and des-(Al-W14)-SHMT
enzymes. pETSH, pSNl and pSN2 plasmids were transformed
into BL21 (DE3) pLysS strain. A single colony was inoculated

374 Reddy Jagath et al. ( E m J. Biochem. 247)
in 50 ml Luria-Bertani medium containing 50 pg/ml ampicillin
and grown overnight at 37°C. 4% of the cells were inoculated
into 1 1 terrific broth media containing 50 pg/ml ampicillin. Af-
ter 3.5 h of growth at 37"C, the cells were induced for 4 h with
0.3 mM isopropyl 1-thio-P-galactopyranoside.The cells were
harvested and the cell pellet was resuspended in 150 ml extrac-
tion buffer (50 mM potassium phosphate, pH 7.4, 5 mM 2-mer-
captoethanol, 1 mM EDTA and 100 pM pyridoxal-P). The cell
suspension was left at room temperature for 1h and sonicated
until it was optically clear. The sonicate was centrifuged at
27000Xg for 20 min. The supernatant was subjected to 25-
65% ammonium sulfate fractionation, blue-Sepharose and Seph-
acryl S-200 column chromatography as described earlier [ll].
The active Sephacryl S-200 fractions were pooled and subjected
to 0-65 % ammonium sulfate precipitation. The pellet obtained
was resuspended in buffer A (50 mM phosphate, pH 7.4, 1mM
2-mercaptoethanol, 1mM EDTA) and extensively dialyzed
against 3 1 of the same buffer (with two changes) for 16h. The
dialyzed sample was used as the enzyme in these studies.
The determination of the isoelectric point (PI value) for
rSHMT, des-(Al-K6)-SHMT and des-(A1-W14)-SHMT
helped us in the design of a new purification protocol for the
rSHMT and des-(A1-Wl4)-SHMT. The new protocol involved
the use of CM-Sephadex instead of blue-Sepharose as reported
earlier [ll]. The crude extracts for rSHMT and des-(A1-W14)-
SHMT were prepared as described before. The crude extracts
were subjected to 0-65 % ammonium sulfate precipitation, the
pellet obtained was resuspended in 20 ml 20 mM potassium
phosphate, pH 6.4, 1mM 2-mercaptoethanol, 1mM EDTA and
50 pM pyridoxal-P and then dialyzed for 24 h against the same
buffer (1 1 with two changes). The dialyzed sample was centri-
fuged at 27000Xg for 10 min and the supernatant was loaded
on to a CM-Sephadex column (2X20cm) which was equili-
brated with 20 mM potassium phosphate, pH 6.4, 1 mM 2-mer-
captoethanol, 1 mM EDTA and 50 pM pyridoxal-P. The column
was washed with same buffer (I 1) and the bound enzyme was
eluted with 100 ml200 mM potassium phosphate, pH 7.4, 1 mM
2-mercaptoethanol, 1mM EDTA and 50 pM pyridoxal-P. The
eluted sample was subjected to 0-65% ammonium sulfate pre-
cipitation. The pellet obtained was resuspended in 4 ml 200 mM
potassium phosphate, pH 7.4, 1 mM 2-mercaptaethano1, 1mM
EDTA, 50 pM pyridoxal-P and subjected to Sephacryl S-200
column chromatography as described earlier [1I]. Fractions con-
taining enzyme were pooled and precipitated at 65% ammonium
sulfate and the pellet was resuspended in buffer A and dialyzed
against 1 1 of the same buffer (with two changes) for 16h and
this enzyme preparation was used in these studies.
Enzyme assay. SHMT activity was assayed using [ 3 - T ]
serine and H,-folate as substrates [29, 301. One unit (U) was
defined as the amount of enzyme catalyzing the production of
1 pmol HCHO . min-' at 37°C. Proteins were analyzed on a
6% non-denaturing PAGE. SHMT activity staining was per-
formed using /I-phenylserine as substrate [31]. Protein was esti-
mated using BSA as a standard [321 for the determination of the
specific activity.
Isoelectric focussing. Isoelectric focussing was performed
on 5% polyacrylamide gel plates between pH 3.5-10, at
2000 V, 50 mA and 30 W according to the manufacturer's in-
structions (Pharmacia Fine Chemicals). The gel was calibrated
with the standard marker proteins (PI 3.5-10). The purified
rSHMT, des-(A1-K6)-SHMT and des-(A1-W14)-SHMT sam-
ples were applied (20 pl of 1.5mg/ml) on the gel. The proteins
were fixed in 10%trichloroacetic acid and stained with Coomas-
sie brilliant blue R-250 after subjecting to isoelectrofocussing.
Circular dichroism. Circular dichroic (CD) measurements
were made in Jasco-J-500 A automated recording spectropolari-
meter. All CD spectra were recorded at 22 22°C in buffer A
using the same buffer as blank. Far-ultraviolet CD studies were
performed at a protein concentration corresponding to 0.1 A2R0
unit/ml (where 1 A,,,, unit is the amount giving an absorbance
of 1 at 280 nm in 1 nil solution in a 1-cm light path). The CD
spectra obtained were plotted as molar ellipticity assuming a
relative subunit molar mass of 52900 g/mol for the rSHMT,
52319 g/mol for des-(Al-K6)-SHMT and 51622 g/mol for
des-(A1-W14)-SHMT. The subunit molar masses were calcu-
lated from their respective amino acid sequences. OM, =
(Bm"XM)/cXd where Omo = observed ellipticity, M = subunit
molar mass, c = concentration of protein in mg/ml and d = path
length in dm.
Visible CD spectra were recorded in a Jasco-J-20 C auto-
mated recording spectropolarimeter. The protein concentration
corresponding to 1 A,,, unit/ml in buffer A was used. The molar
absorption coefficients for rSHMT, des-(A1-K6)-SHMT and
des-(A1-W14)-SHMT were calculated from the amino acid se-
quence by the method of Gill and von Hippel [33] as
168325.2 M-' cm-', 178968.8 M-' cm I, 145320.5M-' cm-',
respectively ; the concentration of these proteins was calculated
using the absorption coefficients.
Inactivationof the SHMTs by heat and urea. rSHMT, des-
(A1-K6)-SHMT and des-(A1-W14)-SHMT (125 pg/ml) were
incubated in buffer B (50mM potassium phosphate pH 7.4,
1mM 2-mercaptoethanol, 1mM EDTA, 50 pM pyridoxal-P) at
50°C or 55°C. The aliquots (10 pl) were withdrawn at 0, 5, 10
and 15 min, cooled to 0°C and assayed for residual enzyme ac-
tivity (301.They were also incubated at 12.5 pg/ml in buffer B
with urea (0-2 M) at 3022°C for 2 h. An aliquot (80 pl) of
this reaction mixture was used directly for enzyme assay by the
addition of ~-[3-'~C]serine(10pl) and H,-folate (10 pl) [30].
Thermal denaturationstudies. The thermal denaturation of
rSHMT, des-(A1-K6)-SHMT and des-(Al-W14)-SHMT was
performed in a Gilford Response I1 spectrophotometer from Ms.
Ciba Corning Diagnostics, as described by Bhaskar et al. [7]
with the following modifications. The protein samples (250 pl
of 0.3 mg/ml) in buffer A were heated from 30°C to 80°C at
the rate of 1"C/min using the software built into the instrument.
The absorbance change in each case was monitored at 287 nm
and data averaged from two experiments. The first derivative of
the thermal denaturation profiles was used to evaluate the appar-
ent transition temperatures of the three proteins. Similar condi-
tions were used to monitor the thermal denaturation profiles of
rSHMT and des-(A1-W14)-SHMT in the presence of serine,
glycine and alanine (I00mM) and pyridoxal-P (50 pM) using
the buffer blank in the reference cuvette with the same ligand
concentration.
Apoenzyme preparation. Apoenzymes of rSHMT, des-
(A1-K6)-SHMT and des-(A1-W14)-SHMT were prepared as
described earlier 1341 with minor modifications. D-Alanine
(200 mM) was added to the holoenzyme (1 mg/ml) in 50 mM
potassium phosphate pH 7.4, 10mM 2-mercaptoethanol, 1 mM
EDTA, 200 mM ammonium sulfate and incubated at 37°C for
4 h. The pyruvate and pyridoxamine 5'-phosphate (pyridox-
amine-P) formed during the reaction were removed by passing
the sample through a 30-kDa Centricon filter.
Size-exclusion chromatography. To determine the native
molecular mass of rSHMT, des-(A1-K6)-SHMT and des-(A1-
W14)-SHMT, a Superose 6 HR 10/30 analytical gel filtration
column was used on a Pharmacia FPLC system. The column
was calibrated with the standard proteins apoferritin (440 kDa),
sheep cytosolic SHMT (213 kDa), yeast alcohol dehydrogenase
(150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase
(29 kDa) and cytochrome c (12.4 kDa). The buffer used for this
study was buffer A containing 0.1 M KCl.

Reddy Jagath et al. (EM .I. Biochern. 247) 375
97.
68-
-97
-68
43 - -43
29- -29
14- -14
Fig. 1. The SDSRAGE analysis of the expressed proteins of N- and
C-terminaldeletion clones of SHMT. Aliquots equivalent to 0.15 and
0.25 A,,,,, units of E. coli cells were used for the analysis of total and
soluble proteins, respectively. Lanes 1 and 24, molecular mass markers;
lane 2, total protein and lanes 3 and 22 soluble proteins of PET-3e; lane
4, total protein and lanes 5 and 23, soluble proteins of pETSH; lanes 6,
8, 10, 12, 14, 16, 18, 20 total proteins and lanes 7, 9, 11, 13, 15, 17, 19,
21 soluble proteins of pSN1, pSN2, pSN3, pSN4, pSN5, pSN6, pSCl
and pSC2, respectively. The gels were stained with Coomassie brilliant
blue R-250.
The molecular masses of the holo, apo and reconstituted ho-
loenzymes of these proteins were determined on calibrated TSK-
G 3000 SW gel filtration column connected to Shimadzu LC6A
HPLC instrument with an on-line ultraviolet detector (SPD
6AV). The tetrameddimer percentages were calculated by using
area normalization method on HPLC-Chromatopac CR6A and
the amount of sample in each peak was expressed as a percent-
age of the total. The column was equilibrated with buffer A
containing 0.1 M KCI.
RESULTS
Constructionand overexpressionof N- and C-terminal dele-
tion clones of SHMT. Using restriction enzyme and PCR-based
methods, plasmids pSNl (A6), pSN2 (414), pSN3 (430), pSN4
(A49), pSNS (ASS), pSN6 (475) representing N-terminal dele-
tions and pSCl (449), pSC2 (4185) of C-terminal deletions in
the cDNA clone of sheep cytosolic SHMT were constructed.
Restriction enzyme digestions and DNA sequencing of these
clones confirmed the presence of appropriate deletion, ribosome
binding site (Shine Dalgarno sequence), start (ATG) and stop
(TAG) codons in all the deletion clones.
As shown in Fig. 1, des-(A1-K6)-SHMT (lane 7) and des-
(Al-W14)-SHMT (lane 9) were present in the soluble extract.
Although the expressed protein in the case of des-(A1-RSS)-
SHMT (lane 14), des-(A1-G7S)-SHMT (lane 16),des-(Q43S-
F483)SHMT (lane 18) and des-(L299-F483)-SHMT (lane 20)
were present in the total extract, it could not be detected in the
soluble fraction (lanes IS, 17, 19 and 21, respectively). These
proteins were present in the insoluble fraction. The insoluble
extracts were also enzymatically inactive. Unlike these mutant
proteins, des-(Al-V30)-SHMT (lanes 10, 11) and des-(Al-
L49)-SHMT (lanes 12, 13)were not present at detectable levels
in any of the fractions and were barely detectable on western
blots.
Protein sequencing.N-terminal amino acid sequencing showed
that deletions of 6, 14, 58 and 75 amino acids had occurred in
the protein expressed by pSN1, pSN2, pSNS and pSN6 clones,
respectively (Table 1). It can be seen that des-(Al-W14)-
SHMT and des-(A1-G7S)-SHMT had two additional amino
acids (Ala-Arg) at the N-terminus compared to their expected
deletions. These additional amino acids were derived from the
vector during subcloning as indicated by DNA sequencing of
the pSNl and pSN6 clones.
Enzyme purification and characterization.rSHMT, des-(A1-
K6)-SHMT and des-(A1-Wl4)-SHMT were purified as de-
scribed [Ill with the following modifications in the use of E.
coli strain and growth medium. In this study, BL21(DE3) pLysS
strain instead of BL21(DE3) was used [1I] as this strain facili-
tated easy disruption of E. coli cells due to the presence of lyso-
zyme. The terrific broth medium instead of Luria-Bertani me-
dium was used for growing E. coli. The cells grown in 1 1 terrific
broth yielded 40-SO mg purified protein compared to only
10 mg protein from 1 1 Luria-Bertani medium. As shown in
Fig. 2B, compared to rSHMT, des-(Al-K6)-SHMT had a
greater mobility and des-(A1-W14)-SHMT a lower mobility
towards the anode on non-denaturing PAGE. The change in the
net charge on the protein was confirmed by measuring the PI
value by isoeleZtric focussing experiments, the values being
6.85, 5.85 and 7.12, respectively. The information on the PI val-
ues facilitated the standardization of the new protocol using CM-
Sephadex at pH 6.4 for the purification of rSHMT and des-
(A1-W14)-SHMT. The enzymes thus purified were homogen-
eous on non-denaturing PAGE, SDSPAGE (Fig. 2) and amino
acid sequencing. des-(A1-K6)-SHMT and des-(A1-W14)-
SHMT were able to perform H,-folate-dependent aldol cleavage
of serine and H,-folate, independent reactions with p-phenyl-
Table 1. N-terminal amino acid sequence of the deletion mutants of sheep liver cytosolic SHMT.
Protein N-terminal amino acid sequence
rSHMT
Des-(Al-K6)-SHMT
Des-(A1-W14)-SHMT
Des-(A1-R5X)-SHMT
Des-(A1 -G75)-SHMT
MPVNKAPRDADLWSLHEK .....RAVLE . . . . .EGYPGQ . . . . . . . . .
1 I 15 59 76
APRDADLWSLHEK . . . . .RAVLE . . . . .EGYPGQ . . . . . . . . . .
I 15 59 I6
ARSLHEK . . . . .RAVLE .....EGYPGQ . . . . . . . . . .
15 59 76
AVLE . . . . .EGYPGQ . . . . . . . . . .
59 I6
ARYPGQ . . . . . . . . . .
76

376 Reddy Jagath et al. (Eul: J. Biochern. 247)
A 1 2 3 4 k D a B 1 2 3 c 1 2 3
-97
- 68
-43
-29
-14
Fig.2. SDSRAGE and non-denaturing PAGE analysis of rSHMT, des-(Al-K6)-SHMT and des-(Al-W14)-SHMT. The proteins were purified
as described in the ExperimentalProcedures. (A) SDSPAGE analysis.Lane 1, des-(Al-W14)-SHMT; lane 2, des-(Al-K6)-SHMT; lane 3 rSHMT;
all 30 pg. The non-denaturing PAGE of rSHMT (50 pg; lane l), des-(Al-K6)-SHMT (50 pg; lane 2) and des-(Al-W14)-SHMT (45 pg; lane 3)
in duplicate lanes was performed in 6% gels at 4°C for 30 h using 100 V current. A part of the gel (B) was stained with Coomassie and a second
part (C) of the gel was stained for the enzyme activity as described in the Experimental Procedures.
0 5 10 15 20 0 5 10 15 20
Time (min) Time (min)
Fig.3. Thermal inactivation of rSHMT (O),des-(Al-K6)-SHMT (W) and des-(Al-W14)-SHMT (A). The three proteins (125 pglml) were
incubated in buffer B at 50°C (A) or 55°C (B) for 5, 10, 15min. Aliquots (10 pl) were withdrawn and immediately cooled to 0°C. The residual
enzyme activity was assayed [30]. Activity at 0 time was normalized to 100%.
serine and D-alanine as substrates. The enzymes were capable of
forming 343-nm-absorbing geminal diamine, 425-nm-absorbing
external aldimine and 495-nm-absorbing quinonoid spectral in-
termediate species. Des-(A1-K6)-SHMT (0.9 mM, 0.95 mM)
and des-(A1-W14)-SHMT (0.9 mM, 0.9 mM) had similar K,
values for serine and H,-folate, respectively, compared to
rSHMT (0.9mM, 1.0mM). They also had similar specific activ-
ities, i.e. 5.2, 5.5 and 4.9 units/mg, respectively. There were no
marked differences in the secondary structure between the three
proteins as monitored by CD spectra in the range of 195-
250 nm.
Oligomeric structure. rSHMT, des-(A1-K6)-SHMT and des-
(A1-W14)-SHMT proteins were subjected to chromatography
on a Superose-6 HR 10/30 analytical gel filtration column, as
described in Experimental Procedures and the native molecular
mass of these proteins was calculated from a plot of relative
elution volume versus the log (molecular mass) of standard pro-
tein markers. The values obtained were 225 m a , 213 kDa and
190 kDa, respectively. These results suggested all these enzymes
are homotetramers.
Temperature stability studies. rSHMT, des-(A1-K6)-SHMT
and des-(A1-W14)-SHMT (125 pg/ml) were incubated at 50°C
and 55°C for 0, 5, 10 and 15min and the residual enzyme activ-
ity was determined. It is evident from Fig. 3A that incubation at
50°C for 15min, resulted in a minimal loss of activity of
rSHMT and des-(A1-K6)-SHMT, whereas des-(A1-W14)-
SHMT lost >50% activity under similar conditions. When the
temperature was increased to 55"C, the latter lost all its activity
(within 5 min), whereas the other two retained more than 80%
of their activity at this time interval, although increasing periods
of incubation at 55°C resulted in a progressive loss of activity
(Fig. 3B).
The thermal denaturation profile of the three enzymes
showed that while rSHMT and des-(A1-K6)-SHMT behaved
similarly, the midpoint of denaturation curve for des-(A1-
W14)-SHMT was at lower temperature. The apparent melting

Reddy Jagath et al. (Eul: J. Biochem. 247)
14.20
HOLO
cI
377
1
HOLO
100
HOLO
80
R.HUO
J
h
& 60
2.
'5
e
* 40
0.0 0.5 1.0 1.5 2.0 2.5
Urea (M)
Fig.4. Effect of urea on the oligomeric structure and activity of
rSHMT (O), des-(Al-K6)-SHMT (W) and des-(Al-W14)-SHMT
(A). All the enzymes (12.5 pg/ml) were incubated in buffer B at urea
concentrations indicated in the figure for 2 h at 30 2 2°C. Aliquots
(80 pl) were withdrawn and assayed for residual activity by addition of
10 pl each of 36 mM ~-[3-'~C]serineand 18 mM H,-folate as described
[30]. The enzyme activity determined in the absence of urea served as
a control and this activity was normalized to 100%.
Table 2. Thermal stability of rSHMT and des-(Al-W14)-SHMT in
the presence of ligand. The apparent melting points were determined
from the first derivative plots of the denaturation curves (see text for
details).
Ligand Concn Apparent melting point
rSHMT des-(Al-W14)-
SHMT
mM "C
56 52
Serine 100 65 56
Glycine 100 58 55
D-Alanine 100 56 51
Pyridoxal-P 0.05 58 54
-
temperatures values calculated from the first derivative plot were
56"C, 55°C and 52°C for rSHMT, des-(Al-K6)-SHMT and
des-(A1-W14)-SHMT, respectively. It was observed that serine
has a significant effect on the thermal stability of SHMT and
shifted the enzyme from an 'open' to a 'closed' form, whereas
other ligands such as threonine, alanine, etc., had no significant
effect [7, 351. it was therefore essential to compare the effect of
the ligands on the thermal denaturation of these three proteins.
Serine (100 mM) enhanced the apparent melting temperature
from 56 to 65°C for rSHMT, whereas it increased only from
52°C to 56°C for des-(Al-W14)-SHMT. Other ligands such as
glycine (100 mM), D-ahine (100 mM) and pyridoxal-P
(50 pM) caused either marginal increase or no change (Table 2).
Denaturation of rSHMT, des-(Al-K6)-SHMT and des-
(Al-W14)-SHMT with urea. Urea-induced inactivation of
these three proteins was performed as described in Experimental
Procedures. Enzyme activity was measured after incubation with
various concentrations of urea (0-2 M). The mid points of the
inactivation for rSHMT, des-(A1-K6)-SHMT and des-(A1-
W14)-SHMT were 1.820.1 M, 1.720.1 M and 1.220.1 M, re-
spectively (Fig. 4).
12 U 16182022 12U 16 182022
ELUTION VOLUME(ml)
12 I416 182022
Fig. 5. Size-exclusion chromatography of holo, apo and reconstituted
holo enzymes of rSHMT (left), des-(Al-K6)-SHMT (middle) and
des-(Al-W14)-SHMT (right). All the enzyme forms (300pg/ml) in
buffer A were chromatographed on a TSK-G 3000 size-exclusion chro-
matography column attached to Shimadzu LC6A HPLC instrument with
an on-line ultraviolet detector (SPD 6AV). The column was equilibrated
with buffer A containing 0.1 M KC1. The same buffer was passed
through the column at a flow rate of 1ml/min. The elution profiles are
shown in the figure. Peak I and peak I1 correspond to tetramer and dimer
of SHMT, respectively.
The role of pyridoxal-P on the oligomeric structure of
rSHMT, des-(Al-K6)-SHMT and des-(Al-W14)-SHMT.
The apoenzymes were prepared by incubating the proteins with
D-alanine (200 mM) for 4 h at 37°C followed by Centricon
filtration to remove pyridoxamine-P and pyruvate formed in the
reaction. All three apoenzymes had no catalytic activity. The
addition of pyridoxal-P (100 pM) to the apoenzymes resulted in
the regain of 96% and 94% activity for rSHMT and des-(Al-
K6)-SHMT, respectively. However, only 22% of the activity of
des-(A1-W14)-SHMT was regained and increasing the concen-
tration of pyridoxal-P to 1mM did not increase the activity of
the reconstituted protein. The oligomeric status of the three holo,
apo and reconstituted holoenzymes were examined using TSK-
G 3000 gel filtration column. As shown in Fig. 5, all three holo-
enzymes were present predominantly in the tetrameric form with
elution volumes of 14.0 ml, 14.2 and 14.7 ml, respectively; how-
ever, the apoenzymes of rSHMT, des-(A1-K6)-SHMT were
present as a mixture of tetramers (75% and 65%, respectively)
and dimers, while the apoenzyme of des-(Al- W14)-SHMT was
present predominantly as a dimer (95%). When pyridoxal-P
(100 pM) was added to all the apoenzymes, rSHMT, and des-
(A1-K6)-SHMT were present as >96% and 92% tetramer, re-
spectively, while des-(A1-W14)-SHMT was only partially
(20%) converted to tetramer.

378 Reddy Jagath et al. (Eur: J. Bicrchem. 247)
10
8
6
0 4
2
0
~~~
350 375 400 425 450 475 500
Wavelength (nm)
10
B I
350 375 400 425 450 475 500
Wavdength (nm1
Fig.6. Visible CD spectra of holo, apo and reconstituted holo en-
zymes of rSHMT (A) and des-(Al-W14)-SHMT (B). The protein con-
centration corresponding to 1 A?,,, unit/ml in buffer A was used for re-
cording spectra in Jasco-J 20C Spectropolarimeter. (-) Holo,
(-. - . -) apo and (---) reconstituted holoenzymes.
Additional evidence for the role of the N-terminal region in
maintaining the tetrameric structure of the enzyme as indicated
by the ability to form a schiff’s base at the active site was moni-
tored by measuring visible CD spectra. It is evident from
Fig. 6 A that the recombinant enzyme has the characteristic CD
spectra with maximum ellipticity at 425 nm. The apoenzyme
gave baseline ellipticity in the region 350-500 nm. Reconstitu-
tion of apoenzyme with pyridoxal-P yielded a holoenzyme prep-
aration with a CD spectrum identical to that of the native holo-
enzyme. However, the apoenzyme of des-(A1-W14)-SHMT
could only be partially reconstituted with pyridoxal-P (Fig. 6B).
DISCUSSION
The mammalian SHMT which is a homotetramer has been
extensively characterized with respect to its catalytic mechanism
[2, 5, 61. However, there is little information available on the
subunit interactions and stability of this tetrameric enzyme.
Earlier in our laboratory, the cDNA for sheep cytosolic SHMT
was cloned, overexpressed in E. coli and the physico-chemical
as well as kinetic properties of the purified protein studied [111.
The availability of this clone facilitated an in-depth examination
of the structure function relationship of the enzyme. As a first
step in this direction, several clones with specified stretches of
amino acids deleted at the N and C-termini were constructed
and these enabled us to assign a role, if any, for these residues
in catalysis, folding and assembly of the subunits of the enzyme.
Using restriction-enzyme-based methods, pSN2, pSN3, pSN4,
Table 3. Properties of apo, reconstituted holoenzymes of rSHMT,
des-(Al-K6)-SHMT and des-(Al-W14)-SHMT. The holoenzymes
had similar specific activities (4-5 units/mg, see text for details), were
essentially in tetrameric form and exhibited characteristic maximum CD
at 425 nm. These values were normalized to 100. The values given in
the table are expressed as a percentage of their respective holoenzyme
values. The three apoenzymes were catalytically illactive (<1% com-
pared to holoenzyme) and had negligible CD at 425 nm.
Enzyme Property Value for
rSHMT des- des-
(Al- (Al-
K6)- W14)-
SHMT SHMT
5%
Apoenzyme tetramer I5 65 <4
dimer 25 35 >95
Reconstituted holo tetramer >96 >92 20
dinier <3 <s 80
visibleCD (42.5 nm) 98 96 20
SHMT activity 96 94 22
pSN6 and pSC2 deletion clones were constructed. pSN1, pSNS
and pSC3 were constructed by PCR method in which Vent DNA
polymerase was used for the PCR amplifications to limit errors
during amplification.
Under the experimental conditions described in Experimen-
tal Procedures, pSNl and pSN2 were able to express soluble
and catalytically active enzymes, similar to pETSH (Fig. 1). The
low level of expression seen with pSN3 and pSN4 could be due
to the possible secondary structure in the mRNA at the ribosome
binding site and start codon [36, 371, or alternatively the deleted
proteins des-(A1 -V30)-SHMT and des-(A1 -L49)-SHMT,
might be unstable and therefore could not be detected on the
SDYPAGE gels. The expressed des-(A1-R58)-SHMT, des-
(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-
F483)-SHMT mutant proteins were present predominantly in the
insoluble fraction (Fig. I). The deletion of 14 N-terminal amino
acids does not affect the enzyme activity, while deletion of a
large number of amino acids from N and C-terminus resulted in
the expression of proteins in the insoluble fraction.
Studies with porcine AAT revealed that the N-terminal re-
gion was involved in the inter subunit interactions [17]. The
availability of the des-(A1-K6)-SHMT and des-(Al- W14)-
SHMT facilitated an examination of the role of amino acids 1-
14 in the subunit interactions and maintenance of oligomeric
structure. Together with rSHMT, the enzymes were purified to
homogeneity with a yield of 40-50 mg/l. The specific activity
of rSHMT (4.9 U/mg) isolated in this study was comparable to
the value reported earlier [Ill. In addition to performing the
physiological reaction, the mutant proteins were capable of
using /I-phenylserine as a substrate (Fig. 2C) thereby demon-
strating their ability to perform H,-folate-dependent as well as
independent reactions.
A unique feature of the mutant enzymes was the significant
change in their electrophoretic mobility on non-denaturing
PAGE (Fig. 2B) and PI values. A comparison of the N-terminal
sequences of the mutant enzymes (Table 1) showed that in des-
(Al-K6)-SHMT one Lys (Lys 6) was deleted increasing the
overall net negative charge, whereas, in des-(A1-W14)-SHMT
one Lys, Arg and two Asp were removed and one Arg was added
due to vector fusion, resulting in an increase in the overall net
positive charge. These results indicated that the N-terminus in

Reddy Jagath et al. (Eur: J. Bioclzern. 247) 379
SHMT contributed to the net charge carried by the enzyme. A
comparison of the retention volumes of rSHMT, des-(A1-K6)-
SHMT and des-(Al-W14)-SHMT (15.0 ml, 15.1ml and
15.2 ml, respectively) suggested that removal of the N-terminus
resulted in a change in the hydrodynamic volume of des-(A1-
W14)-SHMT which is evidently less thermostable than the other
two proteins (Fig. 3). It was observed that the ligands like serine
convert the enzyme from an ‘open’ to a ‘closed’ form. One of
the consequences of this change is the increased thermal stability
of the enzyme. While the thermal stability of the rSHMT
increased from 56°C to 65”C, upon the addition of serine, this
change was only marginal (52°C to 56°C) with des-(A1-W14)-
SHMT. Another criterion employed for the conversion of open
to closed form is the aldol cleavage of serine which is supposed
to occur only in the closed form [35].The K,,, and V,,,, values
of des-(Al-W14)-SHMT were similar to those of rSHMT in-
dicative of the fact that it was as capable as rSHMT in perform-
ing serine aldol cleavage. These observations taken together sug-
gest that des-(A1-W14)-SHMT can be converted from an
‘open’ to ‘closed’ form and the absence of increased thermal
stability in the presence of serine could be due to the deletion
of the N-terminal arm. It is interesting to recall that in the case of
porcine cytosolic AAT, a deletion of 9 amino-terminal residues
resulted in the large decrease in the thermal stability [17]. Dena-
turation with urea is another convenient monitor of stability of
the enzyme and it is evident that the concentration of urea re-
quired for 50% inactivation of the des-(A1-Wl4)-SHMT is low
compared to rSHMT and des-(A1-K6)-SHMT (Fig. 4). The re-
sults strongly implicate the N-terminal 14 amino acids in the
stabilization of the tetrameric SHMT.
Pyridoxal-P has been implicated in the maintenance of the
oligomeric structure of sheep liver SHMT [12]. In view of the
decreased stability of des-(Al -W14)SHMT compared to
rSHMT, it was necessary to examine the relationship between
the N-terminal arm and pyridoxal-P in the stabilization of the
tetrameric SHMT structure. Removal of pyridoxal-P from the
rSHMT and des-(A1-K6)-SHMT holoenzymes resulted in par-
tial dissociation of the tetramer to dimers. Immediate reconstitu-
tion with pyridoxal-P resulted in 90-95% recovery of enzyme
activity and the formation of the tetramer (Fig. 5). The good
correlation between the recovery of the enzyme activity, visible
CD spectra (Fig. 6) and the tetramer concentration strongly sug-
gested that the tetramer with bound pyridoxal-P is the enzymati-
cally active form of the enzyme (Table 3). In the absence of
pyridoxal-P which stabilizes the tetrameric SHMT, des-(A1-
W14)-SHMT dissociated completely to diniers unlike rSHMT
and des-(A1-K6)-SHMT. Addition of pyridoxal-P to apo des-
(A1-W14)-SHMT resulted in reconstitution to active tetrameric
species only partially ( ~ 2 0 % ;Table 3). These results suggest
that amino acid residues 7-14 are probably involved in dimer-
dimer interactions. The presence of charged residues in this
stretch suggests possible ionic interactions. The results presented
in this paper provide evidence for the architecture of mammalian
SHMT as a dimer of dimers and that this oligomeric structure
is stabilized not only by the cofactor (pyridoxal-P) but also by
the N-terminal arm of the protein.
The authors acknowledge the Department of Biotechnology of the
Government of India for the financial support as well as for providing
automated peptide sequencing facility. We thank Mr V. Krishnan for his
help in the determination of N-terminal amino acid sequence of the dele-
tion mutant proteins. The authors are also grateful for the help and advice
of Dr V. Prakash (CFTRI), Mr B. Gopal, Mr Ramesh Kunmr, Mr B.
Venkatesha, Mr K. A. Ganesh, also to Prof. D. N. Rao and Ms. Mira
Sastri for critical reading of the manuscript.
REFERENCES
1. Blakely, R. L. (1955) Biochem. J. 61, 315-323.
2. Schirch, L. (1982) Adv. Enzymol. 53, 83-112.
3. Schirch, L. G. &Mason, M. (1963)J. Biol.Chem. 238, 1032-1037.
4. Usha, R., Savithri, H. S. & Appaji Rao, N. (1994) Biochim. Biophys.
5. Manohar, R. & Appaji Rao, N. (1984) Biochem. J. 224, 703-
6. Usha, R., Savithri, H. S. & Appaji Rao, N. (1992) .J. Bid. G em .
7. Bhaskar, B., Prakash, V., Savithri, H. S. & Appaji Rao, N. (1994)
Biochim. Biophys Acta 1209, 40-50.
8. Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N.,
Savithri, H. S. & Appaji Rao, N. (1989) Biochemistry 28, 9607-
9612.
9. Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N.,
Savithri, H. S. & Appaji Rao, N. (1989) Biochemistry 28, 9613-
9617.
10. Acharya, J. K. & Appaji Rao, N. (1992)J. Biol. Chem. 267, 19066-
19071.
11. Jagath-Reddy, J., Ganesan, K., Savithri, H. S., Asis Datta & Appaji
Rao, N. (1995) EUKJ. Biochem. 230, 533-537.
12. Brahatheeswaran, B., Prakash, V., Savithri, H. S. & Appaji Rao, N.
(1996) Arch. Biochem. Biophys. 330, 363-372.
13. Sandmeir, E. & Christen, P. (1980) J. Biol. Chem. 255, 10284-
10289.
14. Triate, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J.
Bid. Cheni. 259, 723-728.
15. Kim, D. S. & Churchich, J. E. (1983) J. Bid. Chem. 258, 11768-
11773.
16. Schirch, V., Schirch, D., Martini, F. & Bossa, F. (1986) ELMJ. Bio-
chem. 161, 45-49.
17. Fukumato, Y., Tanase, S., Nagashima, F., Ude, S., lkegami, K. &
Morino, Y. (1991) J. Biol. Chem. 266, 4187-4193.
18. McPhalen, C. A,, Vincent, M. G. & Jansonius, J. N. (1992) J. Mol.
Bid. 225, 495-517.
19. Antson, A. A,, Demidkia, T. V., Gollnick, P., Dauter, Z., Von Tersch,
R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan,
E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195-4206.
20. Toney, M. D., Hohenester, E., Cowan, S. W. & Jansoiiius, J. N.
(1993) Science 261, 756-759.
21. Hatefi, Y., Talbert, P. T., Osborn, M. J. & Huennekens, F. M. (1959)
Biochem. Prep. 7,89-92.
22. Studier, F. W. & Moffatt, B. A. (1986) J. Mol. Bid. 189, 113-
130.
23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989)Molecular don-
ing: a laboralory manual, 2nd edn, Cold Spring Harbor Labora-
tory, Cold Spring Harbor NY.
Acta 1204, 75-83.
707.
267, 9289-9293.
24. Alexander, D. C. (1987) Methods Enzymol. 154, 41-64.
25. Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S., Dunn, J. J. &
26. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Nati Acad.
27. Laemmli, U. K. (1970) Nature 227, 680-685.
28. Matsudaira, P. (1987) J. Bid. Chem. 262, 10035-10038.
29. Taylor, R. T. & Weissbach, H. (1965) Anal. Biochem. 13, 80-84.
30. Manohar, R., Ramesh, K. S. & Appaji Rao, N. (1982) J. Bioscr. 4.
31-50.
31. Ulevitch, R. J. & Kallen, R. G. (1977) BiochemisfT 16, 5342-
5349.
32. Lowry, 0.H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951)
J. Biol. Chem. 193, 265-275.
33. Gill, S. C. & von Hippel, P. H. (1989) Anal. Biochem. 182, 319-
326.
34. Schirch, D., Fratte, S. D., Iuresia, S., Angellaccio, S., Contestabile,
R., Bossa, F, & Schirch, V. (1993) J. Biol. Chenz. 268, 23132-
23 138.
35. Schirch, V., Shostak, K., Zamora, M. & Gautam-Basak, M. (1991)
J. Biol. Chem. 266, 759-764.
36. Gold, L. (1988) Annu. Rev. Biochem. 57, 199-233.
37. Hall, M. N., Gabay, J., Debarbouille, M. & Schwartz, M. (1982)
Studier, F. W. (1987) Gene 56, 125-135.
Sci. USA 74, 5463-5467.
Nature 295, 616-618.

rSHMT_EJB_1997

More Related Content

What's hot (20)

Viewers also liked (14)

Similar to rSHMT_EJB_1997 (20)

rSHMT_EJB_1997