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INTRODUCTION |
5-Aminolevulinate synthase (ALAS)1 (EC 2.3.1.37)
catalyzes the condensation of glycine and
succinyl-CoA to form 5-aminolevulinic acid (ALA), CoA, and carbon
dioxide. This is the first and the major regulatory reaction in the
heme biosynthetic pathway in non-plant eukaryotes and the
-subclass
of purple bacteria (1, 2). Mammals encode two distinct ALAS isoforms,
housekeeping ALAS and erythroid-specific ALAS isoform; the latter is
only expressed in developing erythrocytes (3, 4) and is responsible for ~90% of the total synthesized heme in the body. The gene encoding the human erythroid ALAS has been localized on the band Xp11.21 of the
X chromosome (5), whereas the gene for the human housekeeping ALAS has
been assigned to the band 3p21 of chromosome 3 (6). Mutations in the
human erythroid ALAS have been associated with X-linked sideroblastic
anemia (7-10), a disorder characterized by inadequate formation of
heme and an overaccumulation of iron in erythroblast mitochondria
(7).
Murine erythroid ALAS functions as a homodimer, with the active site
residing at the subunit interface (11), and requires pyridoxal
5'-phosphate (PLP) as an essential cofactor (2). In murine erythroid
ALAS, the PLP cofactor is covalently bound to the Lys-313 residue
through a Schiff base linkage, forming the cofactor-protein complex
termed internal aldimine (12). In addition, the conserved
Lys-313 residue was reported to have a catalytic role (13, 14).
Steady-state kinetic analysis of the ALAS-catalyzed reaction indicated
an Ordered Bi Bi mechanism, in which glycine binds before succinyl-CoA
and ALA is dissociated from the enzyme last (15). The chemical
mechanism of the ALAS reaction appears to be similar to that of other
PLP-dependent enzymes catalyzing reactions involving amino
acids (16, 17). Briefly, the binding of glycine leads to the formation
of the PLP-glycine complex termed external aldimine, which
upon removal of the pro-R proton of glycine yields a
resonance-stabilized quinonoid intermediate. Subsequently, this intermediate reacts with the second substrate succinyl-CoA forming a
putative
-amino-
-ketoadipate aldimine (14). The next steps involve the decarboxylation of the glycine-derived carboxyl group and
formation of an aldimine to ALA. The release of ALA, or a protein
conformation change associated with it, was suggested to be the
rate-limiting step of the ALAS-catalyzed reaction (18).
Although the three-dimensional structure of ALAS has yet to be
determined, the evolutionary proximity between ALAS and other members
of the
-family of PLP-dependent enzymes of known
three-dimensional structure made it possible to perform homology
modeling studies of ALAS structure and function (17). The Arg-439
residue is involved in the binding of glycine by forming a salt bridge
with its negatively charged
-carboxylate group (19). The Asp-279 residue appears to be positioned close to the pyridinium ring nitrogen
of the PLP cofactor. Its negatively charged carboxylate group
stabilizes the protonated form of the ring nitrogen, thus enhancing the
electron sink capacity of the PLP cofactor (20). Tyr-121 has been shown
to be involved in PLP cofactor binding by donating a hydrogen bond from
its hydroxyl group to the phosphate oxygen of the PLP cofactor
(21).
Whereas the roles of defined active site amino acids in the structure
and catalytic mechanism of ALAS have been recently explored using
site-directed mutagenesis (14, 19-21), much less is known about the
role of the ALAS polypeptide chain arrangement in folding, final
structure, and catalysis. Circular permutation, which disrupts the
continuity of the polypeptide chain by placing the original N and C
termini at new locations, has proved to be a valuable tool for studying
the effects of polypeptide chain rearrangements on catalytic activity
and folding of proteins (22, 23). With circular permutation of
proteins, the natural N and C termini are covalently linked, and new
termini are created upon cleavage of the circularized protein at a
different sequence position (24, 25). The result is a change in the
primary (i.e. order of the amino acid sequence), secondary,
and possibly tertiary structures (24, 26). To date, the circular
permutation approach has been successfully applied to more than 20 proteins. These include, for example, T4 lysozyme (22), aspartate
transcarbamoylase (27), disulfide oxidoreductase DsbA (28), and
dehydrofolate reductase (26). Circularly permuted variants have been
constructed either by engineering recombinant variants with N and C
termini at selected locations (23, 26, 29-31) or by randomly
generating new termini (27, 28). In addition, circularly permuted
chains have been generated with protein chemical modification methods
involving the covalent linkage of the N and C termini, followed by
hydrolysis of a single polypeptide bond at a different position from
that of the linked original termini (24). The folding of the circularly permuted variants into functional proteins indicated that these variants can achieve a proper conformation for function and suggested that the amino acid sequence, and not the positioning of the N and C
termini, determines the three-dimensional structure (28, 30, 31).
Importantly, the identification of "functional elements," which
have been defined as the smallest continuous sequences required for
catalytic activity (23), makes possible the exploration of
the "architecture of enzyme function." These functional elements, which are spread throughout the primary structure, define the functional active site in a properly folded enzyme (23).
Here, we analyze the role of the ALAS polypeptide chain in relation to
folding and assembly of the holoenzyme. We report that the circular
permutation of the ALAS polypeptide chain affects neither the
folding/assembly of the ALAS subunits into the dimeric holoprotein nor
the activity of the enzyme. The stable and active circularly permuted
ALAS variants, however, appear to have different topologies.
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EXPERIMENTAL PROCEDURES |
Reagents--
The following reagents were purchased as noted:
Sigma, DEAE-Sephacel,
-mercaptoethanol, PLP, bovine serum albumin,
-ketoglutarate dehydrogenase,
-ketoglutarate, NAD+,
thiamine pyrophosphate, succinyl-CoA, HEPES-free acid, aprotinin, pepstatin, leupeptin, phenylmethylsulfonyl fluoride, the bicinchoninic acid protein concentration determination kit and the gel filtration molecular weight markers kit (cytochrome c, carbonic
anhydrase, bovine serum albumin, alcohol dehydrogenase,
-amylase);
Fisher, glycerol, mono- and dibasic potassium phosphate, disodium EDTA dihydrate; IBF Biotechnics, Ultrogel AcA-44; Bio-Rad, sodium dodecyl sulfate-polyacrylamide gel electrophoresis reagents; New England Biolabs, Vent DNA polymerase, BamHI, SalI,
Ecl136 II, T4 DNA ligase, T4 DNA polymerase Klenow fragment,
T4 polynucleotide kinase, and alkaline phosphatase; Promega, RQ1 DNase;
U. S. Biochemical Corp., T7 Sequenase version 2.0; Stratagene Inc.,
PfuTurbo DNA polymerase; Amersham Pharmacia Biotech,
Superdex 200 gel filtration resin; Cybersyn Inc., DNA oligonucleotides.
The Escherichia coli strain HU227 (32) was a gift from Dr.
C. S. Russell (City University of New York), and the E. coli strain LC24 (33) was obtained from the American Type Culture
Collection (ATCC).
Construction of Plasmid pAC9--
The pAC9 plasmid was used as
the vector plasmid in the construction of the random library of
circularly permuted ALAS variants. pAC9 contains the sequence for stop
codons in the three possible reading frames and for three restriction
enzyme sites (SalI, Ecl136 II, and
BamHI). The Ecl136 II site was engineered so that
the blunt 5' and 3' ends of the insert encoding the circularly permuted ALAS variants would be in frame with the ATG codon (located upstream of
the SalI site (34)) and the first TAA of three stop codons sequence, respectively. Two additional nucleotides (downstream of the
first TAA) were also included to generate the two other reading frames
and, thus, to accommodate inserts that would not be in the same reading
frame as the first TAA codon (Fig. 1). The cassette containing the sequences for the three stop codons and the
three restriction enzyme sites was constructed by annealing two
phosphorylated oligonucleotides (STOP1,
5'-TCGACAGAGCTCTAAATAAATAAG-3', and rSTOP2,
5'-GATCCTTATTTATTTAGAGCTCTG-3'). Upon phosphorylation and
annealing, the oligonucleotides were subcloned into pGF23 expression
vector (34) previously digested with SalI and
BamHI (Fig. 1). Competent E. coli DH5
cells
were transformed with the ligated DNA by electroporation. The screening
for the correct construct was performed by DNA sequencing according to
dideoxy chain termination method (35).

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Fig. 1.
Design of the pAC9 expression plasmid.
Indicated are the steps performed to yield an expression plasmid
containing a cassette with Ecl136 II as the cloning site and
encoding stop codons in the three possible reading frames (see
"Experimental Procedures" for details). pho A, alkaline
phosphatase promoter; Ampr, ampicillin resistance
gene; ALAS, ALAS encoding sequence. The
underlined nucleotides in pAC9 indicate the inserted
nucleotides to create the correct reading frames, and the
arrow indicates the cloning site.
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Construction of Plasmid pAC10--
The pAC10 plasmid was used to
produce sufficient amounts of ALAS cDNA fragment with engineered
SalI sites at 5' and 3' ends. The SalI sites were
engineered such that, upon circularization of the ALAS cDNA piece,
the original ALAS reading frame would be maintained. The ALAS cDNA
fragment with engineered SalI sites was obtained by PCR
using pGF23 plasmid as a template (34) and subcloned into pGF23 vector,
previously digested with SalI.
Construction of a Random Library of Circularly Permuted
ALAS Variants--
The experimental design entailed several
modifications of the method developed by Graf and Schachman (27) (Fig.
2). Briefly, a cDNA fragment encoding
the murine erythroid ALAS with engineered SalI sites at both
the 5' and 3' ends was obtained by digestion of the pAC10
plasmid with SalI, followed by gel purification. The
circularization was accomplished in a final volume reaction of 50 µl
using T4 DNA ligase (8.0 units/µl) and 11 µg of DNA in ligase
buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 10 mM dithiothreitol, 1 mM
ATP, 25 µg/ml bovine serum albumin). Efficiency of the ligation reaction was estimated by agarose gel electrophoresis. Upon heat inactivation of ligase, RQ1 DNase was added at a ratio of 0.002 units
per µg of DNA, and the samples were incubated for 15 min at 16 °C.
The samples were pooled, and the DNA was recovered by phenol
extraction. The generated, linearized inserts were repaired, blunt-ended with Klenow DNA polymerase I fragment, and subcloned into
pAC9 vector (Fig. 1) previously digested with Ecl136 II. Competent E. coli HU227 cells, which can only grow in a
medium containing ALA or when harboring an ALAS expression plasmid,
were transformed with the ligation reaction by electroporation.
One-eighth of the transformation solution was plated onto permissive
2× YT-agar medium (1.6% bacto-tryptone, 1% bacto-yeast extract,
0.5% NaCl, 1.5% agar) containing 50 µg/ml ampicillin and 10 µg/ml
ALA, to score the total number of colonies produced. The remaining
transformation solution was plated onto selective 2× YT-agar medium
containing 50 µg/ml ampicillin, to select only for active ALAS
variants. DNA sequencing templates were prepared from colonies
recovered on the selective medium, and the DNA sequences of the 5' and
3' ends of the fragments encoding the functional circularly permuted variants were determined by the dideoxy chain termination method (35).

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Fig. 2.
Experimental strategy for construction of
circularly permuted ALAS variants-containing library. A murine
erythroid ALAS cDNA obtained by digestion of pAC10 vector with
SalI (see "Experimental Procedures") was circularized,
and the circularized DNA was subsequently treated with RQ1 DNase. The
randomly linearized ALAS-encoding fragments were repaired and
blunt-ended with T4 DNA polymerase Klenow fragment and subcloned into
the pAC9 expression vector to yield a library of circularly permuted
ALAS variants (see "Experimental Procedures" for details).
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Rescue of Non-perfectly Permuted ALAS Variants--
The repair
of staggered ends created after DNase I treatment can lead to the
deletion or insertion of nucleotides, which consequently can yield
extra amino acids, and thus non-perfectly permuted polypeptide chains
(27). To correct the 5' and 3' ends, the fragments encoding these
variants were PCR-amplified using primers coding for the expected 5'
and 3' sequences. The PfuTurbo DNA polymerase was used to
minimize nonspecific incorporation of nucleotides at the 5' and 3' ends
of the amplified inserts, and the PCR conditions were according to the
manufacturer's instructions. The obtained inserts were ligated
into pAC9 vector, previously digested with Ecl136 II.
Competent E. coli HU227 cells were transformed with the
ligation, and the 5' and 3' end insert sequences of the functional rescued variants were verified by dideoxy DNA sequencing (35).
Construction of Rationally Designed Circularly Permuted ALAS
Variants--
Two ALAS cDNAs were ligated in tandem and in the
original reading frame, using an ALAS expression plasmid, pGF23
(34), as the DNA template for two PCRs. In PCR1, the 5' primer
(NALAS-6, 5'-GCATATGTCGACTGACAGGAAGAGCAAGATTG-3') contains
the sequences for the first six amino acids of the mature ALAS and for
a SalI site (sequence for SalI site is
underlined) and the 3' primer (RALAS-75,
5'-AGCTCAATTGAGCATAGGTGGTAAC-3') contains the sequences for
the terminal six amino acids and for an MfeI site (sequence for MfeI site is underlined). In PCR2, the 5' primer
(MALAS-76, 5'-TGCTCAATTGGACAGGAAGAGCAAG-3') contains the
sequences for the first six amino acids of the mature ALAS and for an
MfeI site (sequence for MfeI site is underlined),
and the 3' primer (CALAS-7, 5'-GCAGCTGGATCCTTAAGCATAGGTGGTAACAT-3') contains the
sequences for the terminal six amino acids and for a BamHI
site (sequence for BamHI site is underlined). The purified
PCR1 product was digested with SalI and MfeI,
whereas the purified PCR2 was digested with BamHI and
MfeI. The digested PCR products were then ligated into pGF23
previously digested with SalI and BamHI. The
generated plasmid, pAC1, encoding the two ALAS cDNAs in tandem was
subsequently used as the PCR template in the construction of circularly
permuted variants of designed N and C termini. The 5' and 3' primers
were 5'-TATAGTCGACTGCTGCATGCAACTTCTGTA-3' and
5'-TATAGGATCCTTACACAGACACATCTTGGAA-3', respectively, for A475 variant;
5'-TATAGTCGACTATGGAAAACTTTGTGGAGA-3' and
5'-TATAGGATCCTTACATCTGAGGGCTGTGAAA-3', respectively, for M451 variant;
5'-TATAGTCGACTTCTGGGGCTCTAGAATCTA-3' and
5'-TATAGGATCCTTAGAGCATCATGGGAGGCAG-3', respectively, for S351 variant;
5'-TATAGTCGACTCAGTATGGAGCCCTGACCT-3' and
5'-TATAGGATCCTTAGTGGGCCACATCACACAA-3', respectively, for Q271 variant;
5'-TATAGTCGACTAAGTTTCATGTGGAGCTT-3' and
5'-TATAGGATCCTTAGCTGGTACCTGAGATATT-3', respectively, for K156 variant;
and 5'-GCATATGTCGACTGGTTATGACCAATTTTTCA-3' and
5'-TATAGGATCCTTAGAAAGCCTGGCTTCCAGT-3', respectively, for Y167 variant.
Purification of Selected Circularly Permuted ALAS
Variants--
Recombinant wild type and circularly permuted ALAS
variants were purified from E. coli overproducing cells
containing the ALAS-encoding cDNAs under the control of the
alkaline phosphatase (pho A) promoter (34). E. coli strain BL21(DE3) cells harboring the expression plasmids for
Q69, N404, and N408 ALAS variants or E. coli strain LC24
cells (33) harboring the expression plasmid for L25 were grown in MOPS
medium and harvested as described previously (34). Cell pellets were
resuspended as in Ref. 34, with the exception of the pellet
corresponding to the BL21(DE3) cells harboring the N404 variant, which
was resuspended in 20 mM potassium phosphate buffer, pH
7.5, containing 5% glycerol, 1 mM EDTA, 20 µM PLP, 5 mM mercaptoethanol, and the
following protease inhibitors: 1 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, and 1 mg/ml phenylmethylsulfonyl
fluoride. The steps following cell lysis and centrifugation were
essentially as described in Ref. 34 with slight modifications.
Specifically, the initial ammonium sulfate fractionation step was 20%
for the Q69 variant, 25% for the N408 and L25 variants, and 28% for
the N404 variant. After stirring for 10 min at 4 °C, the solution
was centrifuged at 27,000 × g for 30 min at 4 °C,
and the supernatant was further fractionated with ammonium sulfate to a
final concentration of 30% (in the case of L25 and Q69 variants) or
38% (in the case of N408 and N404 variants). The chromatographic steps
using an Ultrogel AcA and DEAE-Sephacel columns were as described
previously (34), with the following modifications: the DEAE-Sephacel
resin was washed with Buffer A until A280 was
lower than 0.1 and the protein of interest (i.e. wild type
or circularly permuted ALAS variants) was eluted with Buffer A (34)
containing 60 mM KCl. Protein-containing fractions were
pooled and concentrated in an Amicon 8050 stirred cell with an YM30
membrane. The purified and concentrated enzyme (wild type or variants)
was stored under liquid nitrogen.
Protein Concentration Determination, Native PAGE, and
SDS-PAGE--
Protein concentration was determined by the
bicinchoninic acid assay, according to the instructions supplied with
the protein concentration determination kit, and using bovine serum
albumin as standard. Protein purity was assessed by SDS-PAGE (36). The oligomeric state of the rationally designed ALAS variants was verified
using native PAGE (37). The protein samples were prepared from E. coli cells, harboring the different expression plasmids, which
were induced for protein expression and grown for 16 h at 37 °C
(34). Cells were harvested and lysed using sonication, and the cell
membranes were pelleted. Most of the supernatant protein was
precipitated by addition of a saturated solution of ammonium sulfate to
a final concentration of 50%. The precipitated protein was resuspended
in 300 µl of 20 mM potassium phosphate buffer, pH 7.5, containing 15% glycerol. The resuspended protein was desalted using
Bio-Rad Bio-Spin 6 gel filtration columns, and ~30 µg of the
desalted protein samples were loaded onto the gel.
Molecular Mass Determination of ALAS Variants by Gel Filtration
Chromatography--
The native molecular masses of ALAS variants were
determined by gel filtration chromatography on Superdex 200 column
(1.0 × 50 cm). The Superdex 200 gel filtration column, which was
connected to a PerkinElmer Life Sciences high pressure liquid
chromatography system, was equilibrated with 20 mM
potassium phosphate buffer, pH 7.5, containing 10% glycerol, 1 mM EDTA, 20 µM PLP, and 5 mM mercaptoethanol. The flow rate was set at 1 ml/min. The gel filtration molecular weight markers and the purified ALAS variants were dissolved in the same buffer and applied onto the column under the same conditions. The molecular masses of the ALAS variants were calculated using the linear equation obtained from the calibration curve, which
was generated from the molecular weight markers.
UV-visible Absorption and CD Spectrocopies--
Shimadzu UV2100U
UV-visible dual beam spectrophotometer was used to obtain all
UV-visible absorption spectra. This spectrophotometer is equipped with
thermostatically controlled cell holders and allows exporting data as
ASCII files through the RS232 interface. CD spectra were obtained on a
Jasco model 710 spectropolarimeter calibrated for both wavelength
maxima and signal intensity using an aqueous solution of
D-10-camphorsulfonic acid (38). CD spectra (6-12
µM enzyme) were recorded over the wavelength range
200-270 nm using a cylindrical cell of 0.1 cm path length and a total volume of 300 µl. The observed rotation degrees (
obs)
were converted to molar ellipticity. All spectra were obtained at
25 °C and corrected for buffer contribution.
Determination of Glycine Dissociation Constant
K
--
The glycine dissociation
constants for wild type ALAS and its variants were determined by
spectrophotometrically titrating the proteins in 20 mM
potassium phosphate, pH 7.5, containing 10% glycerol, 1 mM
EDTA, and 5 mM mercaptoethanol with glycine at 20 °C.
UV-visible spectra were recorded from 250 to 500 nm after each addition
of glycine to the protein of interest. High concentration glycine stock
solution (2 M) was used to ensure minimal dilution of the
protein sample. The binding of glycine to the enzyme was determined by
monitoring the increase of absorbance at 410 nm, which corresponds to
the formation of external aldimine between PLP and glycine (19). The
obtained absorbance change data were fit to the Equation 1,
|
(Eq. 1)
|
where Y is the ratio between the absorbance increase
at 410 nm (
A410) and the maximal absorbance
increase at 410 nm (
Amax), and
[Gly]tot is the total glycine concentration.
Steady-state Kinetic Characterization of ALAS Variants--
The
steady-state kinetic parameters
K
,
K
, and
kcat of ALAS and ALAS variants were determined
at 20 °C using a continuous spectrophotometric assay as described
previously (39). To determine the apparent Km
(K
) and maximal velocities
(V
) the data were analyzed in
matrices of six glycine and six succinyl-CoA concentrations and fit to
Equation 2.
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(Eq. 2)
|
To determine the Km and Vm values,
the apparent K
and
V
were fit to Equations 3 and
4,
|
(Eq. 3)
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(Eq. 4)
|
where K
and
K
are Michaelis constants
for glycine and succinyl-CoA, respectively, and
K
is the limiting value of
K
when glycine
concentration approaches zero.
Modeling of the Three-dimensional Structure of the Wild Type ALAS
and Circularly Permuted ALAS Variants and Prediction of Secondary
Structure of the Wild Type ALAS--
The primary structure and the
x-ray structure coordinates of the E. coli
8-amino-7-oxononanoate synthase (AONS) (40, 41) (Protein Data Bank
accession code 1BS0) were used to model both the wild type ALAS and its
circularly permuted variants. Molecular modeling was performed using
the automated protein modeling server Swiss model (42-44). The
First Approach Mode and the lower Blast limit of 0.0001 were used.
Protein topology schematics (TOPS) (45, 46) were generated using
the automated TOPS-generating server. The secondary structure of the
ALAS was predicted using the Garnier (GOR IV) method (47).
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RESULTS |
Construction of a Random Library of Circularly Permuted ALAS
Variants and Screening for Active ALAS Variants--
To evaluate the
role of the ALAS polypeptide chain in folding, assembly of the
holoenzyme, and function, we constructed a library of randomly
circularly permuted murine erythroid ALAS cDNAs using a modified
version of the method described by Graf and Schachman (27). The overall
scheme is depicted in Fig. 2. A circularized ALAS cDNA was
synthesized such that the first and last residues of the natural ALAS
were connected. The major modification of our method in relation to
that described previously (27) was the omission of a sequence encoding
a peptide linker between the natural N and C termini. In fact, as far
as we are aware, this is the first time that circularly permuted
variants have been constructed without a peptide linker between the
natural termini of the protein. Typically, linkers have been introduced so that the two natural termini can be connected without steric strain
(27, 28). The circular ALAS cDNA was randomly linearized upon
digestion with RQ1 and any generated 5'- and 3'-protruding ends were
repaired with DNA polymerase. The blunt-ended inserts encoding the
library of circularly permuted ALAS variants were cloned into the pAC9
expression plasmid (Fig. 1), which was designed to contain an
Ecl136 II cloning site and stop codons in the three different frames, so that inserts that did not terminate in the same
frame as that of ALAS could be accommodated (Fig. 1). To identify
active circularly permuted ALAS variants, the
hemA
E. coli strain HU227 was
transformed with the library, and ~17,500 clones were screened for
their ability to rescue the HU227 cells in media containing neither ALA
nor heme. Of these, 180 transformants were identified as active, based
on the ability to grow on ALAS selection medium (2× YT + ampicillin).
Sequencing of the 5' and 3' ends of 172 active clones indicated that 21 corresponded to circularly permuted variants, whereas the remaining 151 clones encoded variants with the natural ALAS N and C termini. Thus, ~12% of the active ALAS variants were circularly permuted. However, out of the 21 active variants only 9 were identified as different circularly permuted ALAS variants.
Most of the active variants exhibited elongations at their 5' end,
which ranged 10-300 base pairs. In contrast, no variants with
shortened termini were identified. The observed elongations most
probably resulted from the RQ1 DNase treatment followed by the
filling-in of the of staggered ends by the Klenow enzyme. DNase I
produces both blunt and staggered ends. The latter, upon reaction with
the Klenow enzyme, can yield 5'-terminal elongations and 3'-terminal
deletions. Similar cDNA modifications were also encountered
previously in the construction of random libraries (27, 28). Since the
active enzymes with terminal elongations do not correspond to perfectly
circularly permuted ALAS variants, these "defective" variants were
"corrected" by PCR amplification using 5' end primers encoding the
new N termini and excluding the elongations corresponding to amino
acids already present at the C termini. Eight of the nine different
circularly permuted ALAS variants conferred heme prototrophy to the
HU227 cells and thus, according to our selection criterion, were
considered as active, perfectly circularly permuted ALAS variants (Fig.
3A). The removal of the
300-base pair long 5' end extension from the ninth circular permuted
ALAS variant yielded a protein incapable of rescuing HU227 cells
grown on selective medium without ALA. It is possible that the
N-terminal extension was required for folding and/or stability and
enzymatic activity in this particular ALAS variant.

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Fig. 3.
Schematic diagram illustrating the amino acid
sequence of the ALAS circularly permuted variants in relation to wild
type ALAS. Top, white bar
represents the wild type (WT) ALAS sequence with highlighted
amino acids previously reported to be essential for ALAS function (12,
19-21). The sequence numbering for the wild type erythroid ALAS is
indicated at the top, and the amino acid numbering at the
left of each variant indicates its N-terminal amino acid.
Thus, for each variant the amino acid corresponding to the wild type
ALAS N-terminal amino acid is represented by the junction between the
black and white bars. A,
active, perfectly circularly permuted ALAS variants obtained from the
screening of a random library. B, rationally designed ALAS
circularly permuted variants. Only A475 was found to be
active, based on its ability to rescue HU227 cells on selective medium
without ALA.
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The new N termini of the eight active circularly permuted ALAS variants
are mostly centered in protein regions spanning ~100 amino acids from
either the N or the C terminus (Fig. 3A). However, if the
protein regions C-terminal to Lys-313 are considered C-terminal domains, then the swapping of C-terminal domains in front of the natural ALAS N terminus appears to occur to a greater extent (up to
20.8%) than that of N-terminal domains after the natural ALAS C
terminus (up to 13.4%) (Table I). The
new termini of the active, circularly permuted ALAS variants fell both
within predicted secondary structure elements and in predicted loop
regions. That is, the new termini of E13, E16, L25, and E491 variants
(Fig. 3A) are located in
-helices, whereas those of Q69,
N404, and N408 variants (Fig. 3A) are at the border of a
loop and
-helix regions. Finally, D472 had both termini in a
loop region (Fig. 3A). No new termini of active ALAS
variants were identified in central domains, which have been previously
reported to entail active site residues (e.g. Lys-313 and
Asp-279) (14, 20).
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Table I
Occurrence and permutation extent of the active circularly permuted
ALAS variants obtained from the random library
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Rationally Designed Circularly Permuted ALAS Variants--
To
explore the possibility that the termini of the above inactive
circularly permuted ALAS variants had disrupted essential secondary
structural elements essential in folding, dimerization, and stability,
six variants were designed to have their termini spanning the entire
protein sequence and falling outside the sequence for secondary
structural elements (Fig. 3B). Although these six circularly
permuted ALAS variants exhibited similar levels of overproduction in
HU227 cells and appeared to be dimers (data not shown), with exception
of A475, they were inactive as defined by their inability to
rescue the growth of HU227 cells in selective medium without
ALA.
UV-CD, UV-visible Absorption Spectroscopic Characterization and
Oligomeric State of Active ALAS Circularly Permuted Variants--
To
verify whether circular permutations in ALAS introduced substantial
changes in secondary structure, CD spectra in the far-UV region
(200-300 nm) were recorded for the wild type and four active circularly permuted enzymes. As shown in Fig.
4B, the wild type and
circularly permuted ALAS variants displayed similar CD spectra, suggesting that no dramatic changes in the overall conformation and
structural content were introduced.

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Fig. 4.
A, the UV-visible absorption spectra of
purified ALAS circularly permuted variants. The UV-visible absorption
spectra were recorded in 20 mM potassium phosphate buffer
at 20 °C and pH 7.5. Protein samples were 12.5 µM
concentration. B, the normalized UV-CD spectra of purified
ALAS circularly permuted variants. The UV-CD spectra were recorded in
20 mM potassium phosphate buffer at 20 °C and pH 7.5. Protein sample concentrations are as follows: wild type ALAS, 7.4 µM; L25, 6.3 µM; Q69, 6.9 µM;
N404, 5.0 µM; N408, 8.0 µM.
Curves are as follows: ---, wild type ALAS;
···, L25; -··-··, Q69; - - -, N404; -·-·-,
N408.
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Despite possessing the PLP cofactor, the absorption spectra of the
purified, active, circularly permuted ALAS variants differed substantially from that of the wild type ALAS (Fig. 4A). The
major spectral differences arise from the protein-bound PLP cofactor. The wild type ALAS-bound PLP has two distinct absorbance maxima at
330 and
420 nm. With the circularly permuted ALAS variants, these
maxima are shifted toward shorter wavelengths (Fig. 4B), indicating that the local environment of the protein-bound PLP cofactor
might be different in the circularly permuted variants and wild type
ALAS.
To determine the oligomeric state of the native, active circularly
permuted ALAS variants, their molecular masses were determined by gel
filtration chromatography. All of the variants were found to have
molecular masses close to that of the wild type ALAS (
112 kDa (11)).
The small differences probably reflect different Stoke radii between
the circularly permuted variants and the wild type ALAS. Since the
molecular mass of each subunit of the variants and wild type ALAS is
~56 kDa, as determined by SDS-PAGE (Fig. 5), the circularly permuted variants, as
in wild type ALAS, are homodimers. The above findings, taken together,
suggest that circular permutation in ALAS did not prevent folding,
coupling of the PLP cofactor, or assembly of the two subunits into a
functional homodimer.

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Fig. 5.
Determination of the molecular mass of the
circularly permuted ALAS variants by gel filtration
chromatography. Each of the variants ( 6.0 µg) was
independently applied to a Amersham Pharmacia Biotech Sephadex 200 filtration column and eluted with 20 mM potassium phosphate
buffer containing 10% glycerol at 4 °C and pH 7.5 (flow rate 1.0 ml/min). The chromatogram depicted is that of L25 variant. Inset
A, molecular mass calibration curve for the Sephadex 200 column.
The standard proteins used were cytochrome c (12.4 kDa),
carbonic anhydrase (29.0 kDa), bovine serum albumin (66.0 kDa), alcohol
dehydrogenase (150.0 kDa), and -amylase (200.0 kDa). Inset
B, 15% SDS-PAGE of the purified wild type (WT) and
circularly permuted ALAS variants. Approximately 7 µg of each protein
sample were loaded per lane.
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Glycine Dissociation Constants of Wild Type ALAS and ALAS
Variants--
The circularly permuted and wild type enzymes were
titrated with glycine to determine the dissociation constants for
formation of external aldimines with substrate. The titrations were
performed at 20 °C, as some of the circularly permuted variants
(e.g. Asn-408) tended to precipitate at 37 °C. The
dissociation constants (K
) of
the circularly permuted variants ranged from values 2.65-fold smaller
(for N404) to 2.25-fold greater (for Q69) than that of the wild type
ALAS (Table II). Interestingly, the N404
variant exhibited the tightest binding for glycine, with an affinity
increased almost 3-fold over that of wild type ALAS. Previously, we
reported that the Arg-439 residue has an important role in the binding of the carboxylate group of the glycine substrate (19). It is tempting
to speculate that the circular permutation of the ALAS polypeptide
chain in the N404 variant made the glycine-binding domain and the
Arg-439 residue more exposed than in the wild type ALAS and facilitated
the binding of the glycine substrate. In contrast, the Arg-439 residue
might be buried deeper in the protein structure and not readily
accessible to glycine in the L25 and Q69 variants. However, the
circular permutation in the N408 variant increased the
Kd for glycine about 1.3-fold, and the protein
appeared less stable upon glycine binding. It is possible that in this
variant the integrity of the glycine-binding domain might have been
disrupted despite the exposure of the Arg-439 residue. These results
indicate that the C-terminal domain of the protein, which contains the
Arg-439 residue, might mediate efficient glycine binding. The
disruption of this domain and/or relocation of this domain into a more
secluded environment inside the protein affect/s the glycine binding
properties of the ALAS variant. However, at the primary structure
level, this domain can be moved as a whole into the opposite terminus
of the protein without affecting the glycine binding ability of the
enzyme.
Steady-state Kinetic Characterization of Circularly Permuted ALAS
Variants--
To investigate the effects of circular permutation on
the catalytic properties of ALAS, the steady-state kinetic parameters kcat,
K
, and
K
were determined (Table
II). All of the circularly permuted variants exhibited enzymatic
activities either comparable to, or even higher than, that of wild type
ALAS (Table II). The circular permutation in the N404 variant increased
the kcat value ~3.5-fold and increased the
catalytic efficiency for glycine 10-fold. The catalytic efficiency for
succinyl-CoA was increased ~2-fold. The L25 and N408 variants displayed similar kcat values (i.e.
1-2-fold) to that of the wild type enzyme, although the
Km and the catalytic efficiency for succinyl-CoA
were increased ~4-fold in the L25 variant. The increase in the
overall enzymatic performance of some of the variants probably reflects
improved catalytic efficiency toward the substrates (N404 variant for
glycine, N408 variant for succinyl-CoA, and L25 variant for both
substrates). The steady-state kinetic parameters for the Q69 variant
were not determined, due to technical limitations associated with
deviations of the kinetic mechanism from the ALAS Ordered Bi Bi kinetic
mechanism (2). However, neither substrate nor product inhibitions were
identified (data not shown).
Modeling of Wild Type ALAS and Its Circularly Permuted
Variants--
To gain insight into how the circular permutation of the
ALAS polypeptide chain could possibly affect its three-dimensional structure, homology modeling for the structures of the wild type ALAS
and its variants was performed. The high degree of the amino acid
sequence similarity between wild type ALAS and AONS allowed us to
predict the three-dimensional structures of ALAS and its circularly
permuted variants (Fig. 6). The sequences
used in the models for the polypeptide chains of ALAS and its variants
were at least 36% homologous to the template AONS sequence. The major fitness parameters of the models, estimated by the WHAT IF program (48), indicated that the predicted three-dimensional structures were of
good quality (data not shown).

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Fig. 6.
A, the three-dimensional
structure model of the wild type ALAS (monomeric). The superimposed
three-dimensional structures of the wild type ALAS (yellow)
and 7-amino-8-oxononanoate synthase (39) (Protein Data Base code
1BS0) (blue). B, the protein topology schematics
(TOPS) of the predicted three-dimensional structures of wild type ALAS
and circularly permuted ALAS variants. I, wild type ALAS,
E16, L25, D472, and E491 variants. The two possible structural domains
are shown. II, E13 and Q69 variants. III, N404
and N408 variants. The approximate positions of functional amino acid
residues are indicated by arrows. The pointing
triangles represent the -strands "out of" or "into" the
plane of the diagram, respectively; the circles represent
-helices (45). The -strands and the -helices of the /
structure central core are represented in solid black and
patterned black, respectively.
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To simplify the comparison of the structural features of the models,
two-dimensional structure topology schematics (TOPS) were generated
based on the predicted three-dimensional structures (Fig.
6B). Essentially, TOPS are two-dimensional schematic
representations of protein structures. They represent the structure as
a sequence of secondary structure elements and illustrate the relative
spatial position and direction of these elements (46). The unifying feature among wild type and ALAS variants is, as with AONS, the presence of a typical
/
structure with a seven-stranded
-sheet as the central core in which six strands are parallel (Fig.
6B). The wild type ALAS Asp-279 and Lys-313 residues are
located on neighboring
-strands of the central core (Fig.
6B). The structural differences in the predicted
three-dimensional structure models are subtle and appear to reflect a
slightly different arrangement of the secondary structure elements (but
not of their content, as expected from the similar CD spectra).
Specifically, in wild type ALAS and half of the ALAS circularly
permuted variants, the
-sheet central core is surrounded by 9
-helices, with five of the
-helices located on the side of the
sheet facing the active site of the enzyme and the other four on the
side of the
-sheet facing the surface of the protein (Fig.
6B, I). The ALAS circularly permuted variants L25
and Q69 have
-helices equally distributed, albeit less organized, on
both sides of the
-sheet central core (Fig. 6B,
II), and the N404 and N408 ALAS variants display a third
-helical arrangement around the
-sheet central core (Fig.
6B, III). It should be also noted that the
predicted three-dimensional structures of the N404 and N408 variants do
not contain the Arg-439 residue (Fig. 6B, III),
as this protein region was not included in the modeling of the two
variants, given the low BLAST score.
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DISCUSSION |
ALAS is the first PLP-dependent enzyme and the first
enzyme of the tetrapyrrole biosynthetic pathway to be investigated
using circular permutation of the polypeptide chain. The goal of this work was to use circular permutation of the ALAS amino acid sequence to
probe the roles of the natural N and C termini and of the order of the
secondary structure elements in folding and assembly of an active,
dimeric enzyme. Circular permutation of proteins, such as trypsin
inhibitor (24), dihydrofolate reductase (26), aspartate transcarbamoylase (27, 31), E. coli DsbA (28), glucose
transporter (30), Bacillus
-glucanase (49), and spectrin
(50), has revealed that circularly permuted polypeptide chains can fold into stable and active proteins, albeit with, possibly, different topologies from those of the natural proteins. Most of the proteins so
far circularly permuted (slightly over 20) have been small monomers,
which have required linkers connecting the naturally close N and C
termini in order to yield properly folded and active proteins (23, 28,
29, 50, 51). The characterization of active, circularly permuted ALAS
variants, obtained through functional screening of a library of
randomly circularized ALAS variants without linkers between their
termini, has permitted us to conclude that circularly permuted ALAS
variants are capable of folding into functional, native-like
conformations, and thus that the natural location of the termini and
sequential arrangement of the secondary structure elements are not
critical determinants of the final protein fold, PLP cofactor
attachment, or assembly of the two subunits into an active, dimeric enzyme.
The new termini of the active, circularly permuted ALAS variants
interrupted both predicted secondary structure elements and loop
regions, although most of the new termini were centered in 100 amino
acid regions from each of the natural ALAS termini (Fig. 3), indicating
that specific regions of the protein are more tolerant to disruption
(27, 28). The UV CD spectra of the four purified, circularly permuted
ALAS variants are not significantly different from that of the wild
type ALAS, confirming that all of the characterized ALAS variants fold
into an almost identical wild type conformation regardless of the
position of new N and C termini and absence of a polypeptide linker
between the original termini. However, the circularly permuted ALAS
variants exhibited differences in the PLP-binding sites; namely the
UV-visible spectra maxima of the purified variants shifted toward
shorter wavelengths. As with AONS (41), the
425 nm maximum can be
attributed to a planar configuration, in which the electrons of the
double bond between the NZ of the Lys-313 residue and the C4A of the
PLP are conjugated with the delocalized electrons of the pyridine ring.
The 330 nm maximum might reflect the non-coplanar form of the internal
aldimine. The distortion of the PLP-protein complex brought about by
the local environment of the polypeptide chain should interfere with the conjugation and should produce changes in the absorbance spectrum of the protein. The differences in the absorbance spectra of circularly permuted ALAS variants suggest that the local environments within the
active sites of the variants, created by different arrangements of
their polypeptide chains, differ significantly from that of the wild
type ALAS. Significantly, these findings validate the plasticity of the
PLP binding and active site and indicate that as long as ALAS and the
polypeptide chain folds in place for the binding of the cofactor and
for the catalytic residues to be in the correct proximity, then a PLP
fold and an active ALAS are attainable.
The use of the E. coli hemA
HU227 strain (32)
as the initial biological screen, although powerful, had some
limitations. Specifically, the inability of providing heme prototrophy
to this strain could be due to inappropriate folding, proteolytic
degradation, malfunction in PLP binding, or assembly of the two
subunits or could just emulate ALAS variants, which had enzymatic
activities too low to compensate the ALA and heme requirements of the
hemA
strain (32). By studying the active,
circularly permuted ALAS variants (Fig. 3A), as defined
through the biological screen of the random library, we selected for
variants that were biologically functional, with a wild type like fold
and resistant to proteolytic degradation. In contrast, the generation
of six rationally designed circularly permuted ALAS variants (Fig.
3B) permitted us to establish that five of the variants were
dimers, but the lack or the low enzymatic activity (as defined by the
inability to rescue the growth of the hemA
strain) cannot be ascribed, at present, to misfolding, malfunction in
PLP binding, or lowered enzymatic activities.
The comparable specific activities of the purified circularly permuted
variants and wild type ALAS (Table II) suggest tertiary structural
homology of their active sites. However, despite the overall tertiary
structural homology, the arrangement of the secondary structural
elements seems to differ (Fig. 6B). These results led us to
hypothesize that the same ALAS overall structure can be reached through
multiple folding pathways in which adjacent secondary structural
elements interact to form folding units. In fact, unfolding studies of
circularly permuted proteins have demonstrated that, regardless of
their structural similarity to the wild type protein counterpart, their
mechanisms of folding may strongly differ from that of the wild type
(50). Nevertheless, even if multiple protein folding pathways exist,
"there may be essential nucleation sites that are common to all
pathways," as postulated by Hennecke et al. (28). The
identification of such nucleation sites or "modules," which are
compact structural units essential for correct folding and/or
catalysis, can be achieved through circular permutation (23).
Significantly, the identification of the "folding modules" should
reveal the essential features to reach a PLP-binding fold.
The functional mapping of the ALAS polypeptide chain, using circular
permutation clearly, indicates that there are at least two continuous
regions or functional elements (23), which have defined functions in
ALAS catalysis. The first functional element spans from the Phe-70
residue to the Gly-403 residue of the ALAS polypeptide chain. The
integrity of this element is essential for ALAS enzymatic activity, but
it can be moved around the polypeptide chain, without apparently
affecting seriously ALAS function. This functional element contains
residues involved in binding of the PLP cofactor and in enhancing the
properties of the PLP cofactor in catalysis (i.e. Lys-313,
Tyr-121, and Asp-279) (12, 20, 21). We designated this functional
element as the catalytic domain of the ALAS. Curiously, this functional
element is a sub-domain of a previously defined 49-kDa, "core
catalytic domain of erythroid ALAS" (52). The second functional
element appears to span from the Asn-404 residue to the C terminus of
the protein (i.e. Ala-509) and entails the Arg-439 residue,
which was reported to be involved in binding of the glycine substrate
(19). This functional element also can be moved around the polypeptide
chain and disrupted at specific locations without abolishing enzymatic
activity. The relocation of this intact functional element (or glycine
binding domain) to the natural N terminus of ALAS yielded a circularly permuted variant (i.e. N404 variant) with enhanced glycine
binding, enzymatic activity, and catalytic efficiency (Table II). Thus, the change in the order of the functional elements actually improved the function of ALAS. This finding represents a novel view of the
enzyme architecture.
The idea that protein structure can be explained in terms of building
blocks (folding and functional elements or modules) and that the
integrity of these building blocks is essential for folding and enzyme
function has been put forward previously (23). Circular permutation is
a particularly suitable approach for the identification of folding and
functional elements, as the cleavage of the peptide bond within (but
not outside) these elements will prevent folding/function (23). The
identification of two functional elements in ALAS not only supports the
above proposal of functional elements as the "building blocks" of
protein structure but also reveals that the order of these building
blocks is not critical for protein function, as long as the proper
folding of the protein brings them together for catalysis to occur.
Indeed, modeling of the circularly permuted variants indicated a
different arrangement of the secondary structure elements (Fig.
6B), suggesting that a wild type-like tertiary structure can
be achieved through alternative arrangement of the secondary structure
elements of the polypeptide chain. Current research in our laboratory
is aimed at defining whether the ALAS overall structure can be reached
through alternative or multiple folding pathways.