From the Department of Biochemistry, University of
Adelaide, South Australia 5005, Australia and the
Departments of Microbiology and Biochemistry, University of
Illinois, Urbana, Illinois 61801
Received for publication, May 9, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Biotinylation in vivo is an extremely
selective post-translational event where the enzyme biotin protein
ligase (BPL) catalyzes the covalent attachment of biotin to one
specific and conserved lysine residue of biotin-dependent
enzymes. The biotin-accepting lysine, present in a conserved
Met-Lys-Met motif, resides in a structured domain that functions as the
BPL substrate. We have employed phage display coupled with a genetic
selection to identify determinants of the biotin domain (yPC-104) of
yeast pyruvate carboxylase 1 (residues 1075-1178) required for
interaction with BPL. Mutants isolated using this strategy were
analyzed by in vivo biotinylation assays performed at both
30 °C and 37 °C. The temperature-sensitive substrates were
reasoned to have structural mutations, leading to compromised
conformations at the higher temperature. This interpretation was
supplemented by molecular modeling of yPC-104, since these mutants
mapped to residues involved in defining the structure of the biotin
domain. In contrast, substitution of the Met residue
N-terminal to the target lysine with either Val or Thr
produced mutations that were temperature-insensitive in the in
vivo assay. Furthermore, these two mutant proteins and wild-type
yPC-104 showed identical susceptibility to trypsin, consistent with
these substitutions having no structural effect. Kinetic analysis of
enzymatic biotinylation using purified Met Biotin-dependent carboxylases are a ubiquitous family
of enzymes that catalyze the transfer of carbon dioxide between
metabolites using a biotin moiety as a carboxy carrier. These enzymes
play key roles in essential metabolic processes (1). For example, pyruvate carboxylase catalyzes the generation of oxaloacetate, a
precursor for the synthesis of glucose and fat as well as some amino
acids and neurotransmitters (reviewed in Ref. 2). The biotin prosthetic
group is covalently attached via the The structures of two biotin domains have been determined: that of the
Escherichia coli biotin carboxyl carrier protein (BCCP), a
subunit of acetyl-CoA carboxylase (10, 11), and the 1.3 S subunit of
the Propionibacterium shermanii transcarboxylase complex
(TC) (12). These proteins are structurally homologous to the lipoyl
domains of 2-oxo-acid dehydrogenase multienzyme complexes (13, 14),
which undergo an analogous post-translational modification. They have a
flattened Phage display is a powerful technique for investigating the interaction
of proteins and peptides with other macromolecules. Polypeptides are
expressed on the surface of filamentous bacteriophage as fusions to
either the minor (gIIIp) or major (gVIIIp) coat proteins of the virion
(21). The list of proteins that have been displayed on bacteriophage is
ever increasing (see, for example, Refs. 22-24). Recently the biotin
domain of Klebsiella pneumoniae oxaloacetate decarboxylase
was displayed on the surface of We have previously shown that the 104 C-terminal residues of
pyruvate carboxylase 1 (yPC-104) from Saccharomyces
cerevisiae can be expressed as a stable biotin domain in E. coli (7). Furthermore, this peptide can be recognized and
biotinylated in vivo by the E. coli biotin
ligase, BirA. In the present study, we report the expression of
biotinylated yPC-104 on the surface of fd bacteriophage. Using phage
display coupled with an in vivo selection in E. coli, we have investigated regions on the biotin domain molecule
important for the interaction with BPL.
Materials
Oligonucleotides were purchased from Geneworks Ltd (Adelaide,
Australia). The restriction sites in the oligonucleotides are underlined, and mutagenic changes are in bold. The sequences of the oligonucleotides are as follows: YPC104B,
5'-CATACCATGGCAATGAGAAAAATTCGTGTTGCTG-3'; YPC3'B, 5'-
TTACTGCAGACTATGCCTTAGTTTCAACAGGAACT-3'; Lys61Leu, 5'-
AAGATGTGATCATTTCCATGAGCATGGCGCTTAATACGGCTA-3';
YPC104C, 5'-ACAGAATTCCATGGCAATGAGAAAAATTCGTGTTGCTG-3';
YPC-3'C, 5'-GCACAGCACCACCTGCAGACTA-3'.
DNA Manipulations and Sequencing
Polymerase chain reactions, using 100 ng of oligonucleotides
with 10 ng of template, were performed in 40 µl of 1× thermophilic buffer (Promega) containing 2.5-4 mM MgCl2, 2 mM dNTPs, and 1 unit of Taq DNA polymerase with
30 cycles of denaturation at 95 °C for 1 min; annealing at
60-65 °C for 1.5 min, and synthesis at 72 °C for 1.5 min.
Sequencing of all the constructs was performed using either the
Sequenase Version II kit (Amersham Pharmacia Biotech) or ABI Prism Dye
Terminator sequencing (PerkinElmer Life Sciences).
DNA encoding yPC-104 was amplified using PCR with the oligonucleotides
YPC104B and YPC3'B, with template pMW4A, which contains the cDNA
for S. cerevisiae pyruvate carboxylase 1 (26). The primers
introduced NcoI and PstI endonuclease restriction
sites, respectively, facilitating the cloning of the 330-base pair
product into the phagemid pGF-14 (27), a derivative of pHEN-1 (28). This generated the vector pFdBD-104. The biotinylated Lys residue at
position 1135 in pyruvate carboxylase 1 was replaced with a Leu residue
in the construct pFdBD-K1135L. The mutant was obtained by PCR using the
oligonucleotides Lys61Leu and YPC3'B and pFdBD-104 as the template. The
mutated DNA was digested with endonuclease restriction endonucleases
BclI and PstI and ligated into similarly treated
pFdBD-104. Constructs pFdBD-104 and pFdBD-K1135L were transformed into
E. coli XL1-Blue (Stratagene) for phage display.
DNA encoding yPC-104 was cloned into the expression vector pKK223-3
(Amersham Pharmacia Biotech). Oligonucleotide YPC104C was employed,
using PCR with YPC3'B and pFdBD-104, to add an EcoRI endonuclease restriction site onto the 5' end of DNA encoding yPC-104.
The product was digested with EcoRI and PstI and
cloned into similarly treated pKK223-3, generating the construct
pC-104. This vector was subsequently digested with NcoI and
PstI and ligated to the 330-base pair
NcoI/PstI product liberated from pFdBD-K1135L, yielding the construct pC-K1135L. Proteins expressed from these vectors
had an additional Met-Ala motif on the N terminus, as a result of
cloning extra nucleotides at the 5' region of the gene while
introducing the NcoI restriction site.
Expression of gIIIp Fusion Proteins
XL1-Blue cells harboring either pFdBD-104 or pFdBD-K1135L were
grown in 2YT supplemented with ampicillin (100 µg/ml), tetracycline (10 µg/ml), and 10 µM biotin. Overnight cultures were
diluted 1:50 in fresh medium and grown to log phase at 37 °C, after
which the expression of the fusion protein was induced with 0.02 mM IPTG for 16 h at 30 °C. Cell lysates were
prepared as described by Chapman-Smith et al. (8) and
analyzed by SDS-PAGE on 12% polyacrylamide gels (29). Biotinylated
proteins were detected by Western blotting as described previously (8)
and gIIIp fusion proteins detected using anti gIIIp antibody (Mo Bi
Tec, Göttingen, Germany).
Biopanning
Phagemids displaying yeast biotin domains were prepared
essentially as described by Lucic et al. (27). Phagemids
were panned using magnetic beads coated with Streptavidin (Dynal). For
diagnostic panning, 30 µg of beads were incubated with
108 phagemids in 1 ml of PBS, 0.1% BSA for 1 h at
room temperature, captured using a magnet and washed 10 times in 1 ml
of wash buffer (PBS, 0.1% BSA, 0.5% Tween 20). For panning of the
library, 300 µg of beads, preblocked in PBS, 0.1% BSA, 0.025% Tween
20 for 1 h at room temperature, were mixed for 15 min at room
temperature with 1011 phagemid particles equilibrated in
0.5 ml of wash buffer. The magnetic beads were captured and washed 15 times with 1 ml of wash buffer. After washing, beads were resuspended
in PBS, 0.1% BSA and the bead suspension added to log phase XL1-Blue
cells for 15 min at 37 °C. Bacteria infected with phagemids were
detected by growth on LB media supplemented with 100 µg/ml ampicillin.
Construction of Mutagenic Library
DNA encoding yPC-104 was amplified using error-prone PCR.
Oligonucleotides YPC104B and YPC3'C and template pFdBD-104 were employed using the PCR conditions described above, except that dATP was
1 mM and 1 unit of Taq DNA polymerase from a
batch known to be of low fidelity (Promega, batch no. 573 1805) were
included in the reaction mix. PCR products were digested with
NcoI and PstI, and 3.8 pmol of purified insert
was incubated with 0.64 pmol of similarly treated pFdBd-K1135L and 2 units of T4 DNA Ligase (Roche Molecular Biochemicals). DNA was
ethanol-precipitated in the presence of 0.13 M sodium
chloride and 50 µg of carrier tRNA, and then dissolved in 20 µl of
water. Electrocompetent XL1-Blue cells were prepared (30) and
transformed with 1-µl aliquots of the DNA. The transformants were
allowed to recover for 1 h in SOC medium (31) before an aliquot
was withdrawn to estimate the size of the library. Phagemids from the
library were prepared using the method of Gu et al. (32)
with the following alterations; 2YT was supplemented with 100 µg/ml
ampicillin and 10 µM biotin, and expression of the gIIIp
fusion protein was induced with 0.02 mM IPTG. After 1 h of helper phage infection, 50 µg/ml kanamycin was added and the
culture incubated at 30 °C overnight. Phagemid particles were
isolated by precipitation with polyethylene glycol (27) and stored at
In Vitro Biotinylation of Mutant Biotin Domains on Phagemids
Phagemids produced from the library were biotinylated with
purified E. coli biotin ligase (a kind gift from D. Beckett,
University of Maryland, College Park, MD) in buffer containing
40 mM Tris-HCl (pH 8.0), 3 mM ATP, 5.5 mM MgCl2, 50 mM KCl, 5 µM biotin, 0.1 mM dithiothreitol, and 0.1%
BSA. Phagemid particles (0.8-1.2 × 1012
colony-forming units) were reacted with 0.25 pM enzyme at
37 °C for up to 24 h before panning. Phagemids were recovered
from the reaction by precipitation with polyethylene glycol (27) and
the pellet washed to remove free biotin before being resuspended in PBS
for panning.
Analysis of Biotin Domain Mutants
Comparative in vivo biotinylation of the yPC-104
mutants was performed essentially as described previously by Val
et al. (7). The mutants were expressed in E. coli
TM21 cells by IPTG induction for 2 h from the pC-104-derived
expression plasmids described above. Whole cell lysates were prepared
using the method described by Chapman-Smith et al. (8). The
expressed yPC-104 proteins was resolved from total cellular protein by
fractionation on duplicate 15% (w/v) polyacrylamide gels under
denaturing and reducing conditions. Protein was visualized by staining
with Coomassie Brilliant Blue R250 and biotinylated protein detected by
avidin alkaline phosphatase blot (8). The intensity of both the
biotinylated band and the yPC-104 protein band were quantitated by
laser densitometry and the extent of in vivo biotinylation,
expressed as biotinylated protein divided by the total yPC-104 protein,
in arbitrary units, for each peptide. This ensured that the
quantitation of the extent of in vivo biotinylation was
unaffected by any variation in expression levels or protein stability
resulting from the introduced mutations.
Kinetic analysis of enzymatic biotinylation was performed using
purified BPL and apo-yPC-104 peptides as described previously (9).
p values were calculated with two-tailed t tests
using GraphPad Prism for MacIntosh (GraphPad Software Inc, San Diego, CA). Peptides were purified from E. coli BL21( Molecular Modeling
Identification of Homologous Structures--
Protein structures
in the Protein Data Bank predicted to have homology to yPC-104 were
identified by the threading method of Fischer and Eisenberg (34) as
implemented on the UCLA structure-prediction server. The protein
structures with compatibility (Z) scores higher than the
confidence threshold of 5.0 ± 1.0 were as follows: the lipoyl
domain of pyruvate dehydrogenase from Azotobacter vinelandii (Ref. 35; IYV, Z = 10.89), the biotinyl domain of
acetyl-CoA carboxylase from E. coli (Refs. 10 and 11; 1BDO,
Z = 9.65), the lipoyl domain of A. vinelandii 2-oxoglutarate dehydrogenase (Ref. 36; GHJ,
Z = 8.25) and the lipoyl domain of pyruvate
dehydrogenase from Bacillus stearothermophilus (Ref. 14;
LAB, Z = 5.79). From the individual sequence
alignments, a consensus sequence alignment of yPC-104 to these proteins
was generated (Fig. 5). Due to the lack of N-terminal
regions homologous to that of yPC-104 (residues 1075-1098) in the
identified structures, only residues 1099-1170 could be modeled.
Secondary structure was predicted for residues 1075-1098 using the
PHDsec program (37).
Homology Model Construction--
Homology models of apo-yPC-104
and holo-yPC-104 residues 1099-1170 were constructed using the
HOMOLOGY module of the INSIGHTII software (version 98.0; MSI, San
Diego, CA). The structures listed above and the coordinates of the
holo-BCCP domain (Refs. 10 and 11; 2BDO) and apo-BCCP domain (11, 38;
3BDO) were analyzed to identify the structurally conserved regions
(SCRs) by iterative fitting of C Refinement of Models--
The two models were further refined by
restrained simulated annealing using the program X-PLOR (40) employing
the CHARMm force field (41). The restraints employed consisted of the
backbone hydrogen bonds from the respective structure upon which the
model was based (HN-O 1.7-2.0 Å and N-O 2.7-3.0 Å) and harmonic
point restraints on the C Phage Display--
The construct pFdDB-104 was generated, as
described under "Experimental Procedures," to express yPC-104 as a
fusion to the gene III coat protein (gIIIp) of the filamentous
bacteriophage fd in the E. coli supE host strain XL1-Blue
(28). The two moieties were fused via a flexible glycine-rich linker
containing an H64A subtilisin BPN' protease sensitivity motif (33).
Analysis of whole cell lysates by anti-gIIIp antibody and avidin blots
showed that fusion protein expression was inducible with IPTG and the polypeptide was biotinylated in vivo by the bacterial BPL,
BirA (data not shown). A second construct, pFdBD-K1135L, in which the codon for the biotinylated Lys was substituted with a codon for Leu,
but otherwise identical to pFdBD-104, was produced to serve as a
negative control in biopanning procedures. Expression of this gIIIp
fusion protein was detected in whole cell lysates, as described under
"Experimental Procedures." However, as expected, avidin blot
analysis showed that this mutant polypeptide was not a substrate for
the in vivo biotinylation reaction (data not shown).
Phagemids displaying the fusion proteins were prepared under conditions
known to allow monovalent display (28), captured using magnetic beads
coated with streptavidin, and used to infect male E. coli
cells as described under "Experimental Procedures." Those phagemids
bound to the beads were mixed directly with E. coli cells
and transduction assayed by growth in the presence of ampicillin. The
efficiency of panning was such that a single panning step produced a
5,000-fold enrichment of the biotinylated yPC-104 phagemids over the
null phagemid, Bluescript. Similarly low recoveries of phagemids
displaying the nonbiotinylated mutant expressed from
Lys1135 Construction of yPC-104 Mutant Library--
Using phage display
and the in vivo selection described under "Experimental
Procedures," we devised a novel method for investigating the protein:
protein interaction between a biotin domain and a BPL. First, a library
of mutant biotin domains were displayed on the surface of
bacteriophage, and those members that were products of enzymatic
biotinylation were specifically captured with streptavidin. This step
enabled the removal of mutations inducing gross structural changes that
abolished biotinylation. Second, an in vivo genetic selection was employed to segregate the remaining library members specifically into a pool of mutants having decreased affinity for BirA.
Briefly, this selection relies on competition by an efficiently
biotinylated overexpressed protein with endogenous BCCP for a limited
pool of free biotin when expressed in a biotin auxotroph. Expression of
mutants that are poor substrates for BirA results in biotinylation of
BCCP, producing functional acetyl-CoA carboxylase and allowing cell
growth. We have previously used this selection to isolate mutations in
the biotin domain of E. coli BCCP having decreased affinity
for BirA (42).
A phage display library encoding mutated yPC-104 was constructed by
error-prone PCR, using Taq DNA polymerase and limiting the
availability of dATP in the reaction, as described under
"Experimental Procedures." After infecting the transformed bacteria
with helper phage, a phagemid library of ~7 × 106
members was generated. To assess the efficiency of mutagenesis, individual PCR products were introduced into a cloning vector for
sequencing. This revealed an average of one point mutation per 312 base
pairs of amplified DNA. A bias of AT to GC mutations was observed, but
this was not exclusive. To permit mutants of yPC-104 with decreased
affinity for biotin ligase to be captured by the panning procedure,
library phagemids were incubated with purified BirA in vitro
before panning (see "Experimental Procedures"). Furthermore,
panning was restricted to two rounds to minimize the possibility of
losing low affinity mutants over multiple rounds. After two rounds of
panning, the phagemids were used to infect XL1-Blue cells and plasmid
DNA was extracted.
Selection of Biotinylation Defective Mutants in Vivo--
DNA
encoding the mutant biotin domains was excised from the phage display
vector and cloned into the expression vector pC-104 for the genetic
selection. The ligation products were introduced into the biotin
auxotroph E. coli strain TM21 (42) and grown on media
containing IPTG, ampicillin and limited biotin for selection of biotin
domain mutants in vivo. Transformants were replica-plated onto both selective and nonselective media for growth phenotype analysis. Approximately 80,000 colonies were screened in this manner,
and a wide range of colony sizes was observed on the selective media.
Since growth under these conditions results from expression of a poorly
biotinylated protein (42), plasmid DNA was isolated from colonies
showing the best growth. Eighteen isolates giving the expected
restriction pattern were further analyzed by DNA sequencing, and point
mutations were detected in all clones. These data (Fig.
1) documented 12 unique single missense
mutations. In addition, three silent mutations and two double missense
mutations were isolated.
Analysis of Biotinylation in Vivo by BirA--
The mutants
isolated using phage display and the in vivo selection were
analyzed to determine the ability of the peptides to function as
substrates for BirA in vivo at 37 °C, as described under
"Experimental Procedures." Since the extent of biotinylation was
determined as a function of the amount of biotinylated protein relative
to total yPC-104 protein for each mutant protein analyzed, our
quantitation of biotinylation was unaffected by any variation in
expression levels or protein stability resulting from the introduced mutations. The extent of biotinylation for each of the mutants was
expressed relative to wild-type yPC-104, quantitated in the same manner
(Fig. 1). With the exception of the His1102
Several mutations were detected in the amino-terminal region of yPC-104
lying outside the structurally conserved biotin domain. Substitution of
Ala1081 with Pro reduced biotinylation to 52% and the
double mutant Arg1083 Temperature Sensitivity of yPC-104
Mutants--
Temperature-sensitive mutations of BCCP have been shown
to affect the conformation of the biotin domain and, as a result, substrate recognition (5, 42). To investigate the possibility that the
amino acid substitutions in yPC-104 induced structural alterations, the
in vivo biotinylation assay was carried out at a lower
temperature for selected mutants (Fig.
2). Substitutions at all positions
investigated, with the exception of Met1134, showed an
increase in the extent of biotinylation at 30 °C. Substrates
Phe1152 Predicted Structure of yPC-104--
To assist in interpreting the
results obtained from in vivo analysis of apo-yPC-104
mutants, we constructed a molecular model of the yeast biotin domain
(Fig. 3). This model was based on the solution structures of apo-BCCP-87 and several known structurally analogous lipoyl domains (see "Experimental Procedures"). We also predicted the structure of holo-yPC-104 modeled upon holo-BCCP, with
the apo- and holo-yPC-104 structures being essentially identical. For
comparison purposes, the first member of the apo-BCCP (3BDO) and
holo-BCCP (2BDO) NMR ensembles were subjected to identical "refinement" protocols. For both yPC-104 and BCCP models, 10 simulations were carried out and the five final models with the lowest
overall energy were retained for analysis (Table
I). All the model ensembles are tight, in
terms of atomic root mean square deviation values and energetics, and
one model is as good as any other. The deviation of model BCCP
coordinates from the starting experimental coordinates is similar to
the root mean square deviation values for the NMR ensembles (for all
backbone atoms: apo-BCCP, models: 1.78 ± 0.03 Å, NMR: 1.57 ± 0.48 Å; holo-BCCP, models: 1.04 ± 0.02 Å, NMR: 1.33 ± 0.24 Å). The BCCP models have markedly improved Ramachandran properties (apo-BCCP, models: 69.6%, NMR: 40.0%; holo-BCCP, models: 74.9%, NMR: 50.5% of residues in the most favored regions). The yPC-104 models also exhibit comparable backbone atom deviations from
their BCCP-based starting coordinates (apo-yPC-104, 1.26 ± 0.04 Å; holo-yPC-104, 1.17 ± 0.04 Å) and have energy values comparable to those of the BCCP models for all terms except the harmonic term and the electrostatic term. The difference in the magnitudes of harmonic terms between the yPC-104 and BCCP models is
interesting and suggests that the BCCP backbone atom positions suit the
yPC-104 models very well. The electrostatics of the BCCP models are
better than that of the yPC-104 models, as expected since the charged
side chain positions in the BCCP models have some experimental basis.
In addition, yPC-104 has a much higher charge density on its surface
and thus the potential for many more unfavorable interactions.
From sequence alignments, shown in Fig.
4, the eight Interpretation of Mutational Results--
We used temperature
sensitivity to assess the structural stability of yPC-104 mutants. The
three residues where Val
The secondary structure of the 23 N-terminal amino acids of
yPC-104, lying outside the structured biotin domain, was predicted using the PHDsec program (37). Residues
Lys1077-Val1087 were predicted to form an
The mutant Phe1152 Kinetic Analysis of Biotinylation in Vitro--
Wild-type yPC-104
and the two temperature-insensitive mutants, Met1134
When the proteins were assayed with yeast biotin ligase under
conditions that were optimal for biotinylation of yPC-104 (Fig. 6,
C and D; Table II), the wild-type yeast biotin
domain was the preferred substrate, with the affinity for BCCP-87 being
10-fold lower, as described previously (9). The Met1134 Trypsin Susceptibility of Met1134 In all biotin-dependent enzymes, the residues
immediately flanking the biotin-accepting Lys are invariantly Met.
Mutation of these residues in both the biotin-accepting subunit of
P. shermanii transcarboxylase (17) and in synthetic peptides
(18) has revealed that these Met residues are required for the
carboxylation of biotin, the first stable intermediate of the biotin
carboxylase reaction. It has been demonstrated that the flanking Met
residues are not essential for biotinylation by BPLs since proteins
having conservative substitutions are still recognized as substrates (6, 16). In the present report, we isolated two classes of mutations to
the biotin domain of pyruvate carboxylase from Saccharomyces cerevisiae by screening a library of randomly mutated polypeptides using phage display and a genetic selection in E. coli that
permits the isolation of peptides with a lowered affinity for bacterial BPL. The first class of mutations isolated contained two distinct, conservative substitutions of the Met residue immediately
N-terminal of the target Lys. One mutant,
Met1134 It is apparent that BPLs recognize and interact with a structured
biotin domain (13, 42). The second class of mutants isolated using our
selection technique mapped to residues some distance from the target
lysine. All mutants analyzed belonging to this class were
temperature-sensitive, suggesting that these amino acid substitutions
induced conformational changes to the folded domain, making it a
compromised substrate at 37 °C. This finding is consistent with our
previous work, which has demonstrated that several mutations located at
a distance from the target lysine of the E. coli BCCP biotin
domain reduce biotinylation as a consequence of structural alterations
(42). To facilitate interpretation of our mutant data, we have produced
a molecular model of yPC-104 based upon known biotin domain structures
(Fig. 3). Our models of yPC-104 differ from that described by
Brocklehurst and Perham (13) primarily due to the availability of
additional experimentally determined structures from which to construct
a more rigorous model of the protein. Using the BCCP coordinates as a
guide allowed us to better model the loop between The mutations obtained in the present study were mapped onto our model
(Fig. 5). All the temperature-sensitive mutations, predicted to induce
conformational changes to the molecule, were at residues implied to be
necessary for defining protein structure. In addition, the molecular
modeling of the yeast biotin domain highlighted some novel properties,
especially in the N-terminal region outside of the
structured domain. This region contains a predicted The BCCP domain contains a protruding "thumb" between strands Biotinylation and lipoylation in vivo by BirA and the two
lipoyl attachment enzymes, LplA and LipB, respectively, occur in a
highly specific manner despite the striking structural similarity of
the accepting domains (19, 20). It has been demonstrated that the thumb
structure of BCCP functions to inhibit lipoylation, as a thumb deletion
mutant of BCCP-87, which still functioned as a substrate for BirA, was
lipoylated in vivo and in vitro by LplA (19). The
lipoyl domains lack the thumb, having three residues which span the
region corresponding to the base of the thumb, but contain an extended
surface loop between The selection procedure we used was established using E. coli BCCP-87 (42). We showed that, despite BirA having a
10-fold higher affinity for its native substrate over yPC-104 in
vitro, high level expression allowed the in vivo
selection to be adapted for mutational analysis of the yeast protein.
Although no wild-type DNA molecules were isolated after selection,
several silent mutations were obtained. Mutations that destabilize
mRNA, reduce translation efficiency, or interfere with protein
stability would all decrease the selection pressure on the host. These
aspects of our selection procedure account for those mutants obtained
in the selection that functioned as wild-type-like substrates in the
in vivo biotinylation assay, since this assay corrects for
variations in expression levels. It should be noted that the relative
affinities of the mutant peptides obtained from the in vivo
and in vitro biotinylation assays are a reflection of the
different assay conditions. In the in vivo assay that
measures accumulated product, factors such as the concentration of
enzyme, ATP, and biotin cannot be controlled. On the other hand, the
in vitro biotinylation assay measures initial velocity under
steady-state conditions using purified components with optimal,
saturating concentrations of ATP and biotin.
The enzymatic reaction catalyzed by BPLs proceeds through two partial
reactions. In the first partial reaction, ATP and biotin bind BPLs in
an ordered manner (9, 45) and the carboxyl group of inert biotin is
activated by the addition of an adenylate group. In the second partial
reaction, nucleophilic attack upon activated biotin by the amine group
of the Lys in the protein substrate results in the transfer of biotin
from the adenylate onto the apo-biotin domain, with AMP behaving as the
leaving group (45). The Lys side chain must be precisely positioned
into the active site of BPL to bring the electron donating nitrogen
close enough to the reactive biotinyl-5'-AMP moiety to permit the
chemical reaction. Thus, the biotin domain functions as a protein
scaffold that presents the biotin-accepting Lys to the BPLs. The
position of the target Lys in the exposed Thr/Val mutant proteins
with both yeast and Escherichia coli BPLs revealed that
these substitutions had a strong effect upon Km
values but not kcat. The Met
Thr mutant was
a poor substrate for both BPLs, whereas the Met
Val substitution
was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of
the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of a specific Lys
residue within the protein (1), in a reaction catalyzed by biotin
protein ligase (BPL1; EC
6.3.4.15). The primary structure of the biotin domain shows a high
degree of homology between a wide range of enzymes and species (3) and
folds into an independent domain. Deletion studies on enzymes from both
prokaryotes (4, 5) and eukaryotes (6, 7) have shown that biotin domains
can be expressed as stable proteins of around 80 residues, which can be
biotinylated both in vivo and in vitro (8).
Furthermore BPLs from various sources have been found to recognize and
biotinylate acceptor proteins from very different sources (1, 9),
suggesting that all biotin domains fold into an essentially common
tertiary structure and that the information required for association
with BPL is present within this conserved structure.
-barrel structure comprising two four-stranded
-sheets
with the N- and C-terminal residues close
together at one end of the structure. At the other end of the molecule,
the biotinyl- or lipoyl-lysine resides on a highly exposed, tight
hairpin loop between
-strands four and five. The biotinyl-lysine is
found in the motif Met-Lys-Met, which is highly conserved in all biotin
domains (3). The precise positioning of the Lys appears to be important
for recognition by BPL, as moving this residue by one position to
either side of its normal position abolishes biotinylation (15).
Additionally, the properties of the amino acid residues immediately
surrounding the target Lys are important. Conservative substitutions of
the flanking Met residues have revealed that these amino acids are not
essential for interaction with BPL (6, 16), but do affect the
carboxylation and carboxyl-transfer reactions of P. shermanii TC (17, 18). However replacement of the Met-Lys-Met
motif of E. coli BCCP with the Asp-Lys-Ala motif
characteristic of lipoyl domains, abolishes biotinylation by the
bacterial BPL, BirA (19). Moreover, direct replacement of the
lipoylation motif with the biotinylation motif in a lipoyl domain is
not sufficient to specify biotinylation (20). It is evident that the
specificity of biotinylation in vivo requires the
interaction of a BPL with a structured protein substrate containing an
exposed, precisely positioned Lys side chain.
phage (25). Biotinylated phage were
captured using avidin, indicating that the products of an enzymatic
reaction can be specifically selected. Using phage display technology,
proteins can be remodeled through mutagenesis and mutants with
desirable phenotypes selected using an immobilized ligand. An
understanding of the residues required for the function of the
displayed protein can then be obtained by determining the primary
structure of the selected mutants.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C in PBS.
DE3) cells
harboring expression plasmids derived from pET16b (Novagen). These
plasmids were constructed by digesting pET16b with NcoI and
PstI and introducing a similarly treated DNA fragment
containing the mutant biotin domain coding regions, excised from the
plasmids isolated from the in vivo selection.
atom positions and
analysis of hydrogen bonding patterns. For NMR ensembles a mean
coordinate set, generated using MOLMOL (39) was used in the comparison
process. The defined SCRs and loops are indicated in Fig. 5. For the
models of holo- and apo-yPC-104, the SCRs and loop 1 coordinates were
modeled on those of holo- and apo-BCCP, respectively. Coordinates for
loop 2 in each model were based on loop 2 of the lipoyl domain of
pyruvate dehydrogenase (Ref. 35; 1IYV).
atoms tethering them to their
positions in the initial models. This allowed the backbone to remain
relatively fixed and the secondary structure to remain intact while
side chain positions in the core of the domain could be optimized to
reduce steric clashes and promote intimate packing. Simulations were
carried out in vacuo with the dielectric constant set to 10 to approximate solvent screening. Coordinate sets were cooled from 2000 to 100 K over 5 ps of dynamics, followed by 500 steps of conjugate
gradient energy minimization.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu phagemids and the null control were
observed. Thus, the interaction of the phagemids with the
streptavidin-coated beads, and their subsequent capture, was specific
for the presence of a biotin group covalently attached to the phagemids
via Lys1135.
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Fig. 1.
In vivo biotinylation assay.
Biotinylation of yPC-104 mutants at 37 °C was analyzed in
vivo in E. coli, as described under "Experimental
Procedures." The sequence of the 104 C-terminal amino acid
residues of yeast pyruvate carboxylase 1 is shown at the
top, with the biotin-accepting lysine residue indicated
( ). The position and amino acid substitution of the mutations
isolated in this study are indicated below the sequence. Two
isolates containing double mutants are represented by # and *. The
graph shows biotinylation of the mutants relative to the
wild-type peptide. Error bars represent the S.E.
of three replicate experiments.
Arg and
His1117
Arg substitutions, mutations in the region of
the molecule proposed to form the structured biotin domain caused a
decrease in biotinylation. As expected, the Lys1135
Leu
substitution abolished biotinylation and was included in the assays as
a negative control. Two mutations were found at the highly conserved
Met1134 residue adjacent to the biotinyl-Lys. Substitution
of this residue with Val or Thr caused similar decreases in
biotinylation to 69% and 74%, respectively, of wild-type levels.
Substitution of Pro at Ser1141 or Ser1162 and
the Ile substitution at Phe1152 gave decreases in
biotinylation from 40% to 65% of wild-type levels. Two bands were
observed on the avidin blot for the latter mutant (data not shown),
suggesting the protein was susceptible to proteolysis by E. coli enzymes in vivo. Substitution of Val residues with
Ala at positions 1148 and 1166 decreased biotinylation to around 50%,
whereas this substitution at position 1116 did not affect biotinylation
in this assay.
Gly/Lys1086
Ile to
82%, whereas Arg1083
Gly alone had little effect.
Individual mutations from the His1102
Arg/Met1107
Thr double mutant, which showed 46% of
wild-type biotinylation, were cloned into separate vectors and analyzed
but neither single mutation had a large effect on biotinylation (Fig.
1) suggesting that the large decrease observed for the double mutant
was the result of synergistic effects.
Ile and Val1166
Ala were
comparable to the wild-type peptide at the lower temperature. Furthermore, the proteolytic product of Phe1152
Ile
observed at 37 °C was not evident at 30 °C. The efficiency of
biotinylation increased by 31% for Val1148
Ala, 40%
for Ser1141
Pro, and 67% for Ser1162
Pro. We conclude that point mutations of yPC-104 that were temperature-sensitive most likely altered the conformational stability of the biotin domain, rendering it a less favorable substrate for BirA
at the higher temperature. In contrast, substitution of
Met1134 with either Val or Thr were not
temperature-sensitive mutations. The extent of biotinylation of
Met1134
Thr and Met1134
Val was
essentially the same at both 30 °C and 37 °C, suggesting that
these substitutions did not result in structural alteration.
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Fig. 2.
Temperature sensitivity of yPC-104
mutants. The in vivo biotinylation assay described in
Fig. 1 was carried out at 30 °C (white) and 37 °C
(black). The graph represents biotinylation of
the mutants relative to the wild-type peptide at each temperature.
Error bars represent the S.E. of three replicate
experiments.
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Fig. 3.
Predicted structure of the yeast PC biotin
domain. A schematic stereo diagram of the predicted structure of
the yeast PC biotin domain (white), produced from the
molecular modeling described under "Experimental Procedures," shown
superimposed on the structure of the BCCP biotin domain determined by
NMR (gray). The position of the biotin accepting
lysines at position 1135 or 122, respectively, is shown, and the
location of the additional sequences in the homologous structures
discussed in the text is indicated. The figure was generated using
Molscript, version 2.1.1 (46).
Measures of coordinate deviations, energies, and stereochemical
properties of the models and the NMR
ensembles
-strands of apo-yPC-104
were defined as structurally conserved regions (
1
Leu1101-Ala1105,
2
Val1110-Ile1111,
3
Leu1121-Ile1122,
4
Pro1127-Ser1132,
5
Glu1137-Ser1141,
6
Gly1146-Val1151,
7
Glu1157-Val1159,
8
Asp1163-Leu1168). Like other biotin and lipoyl
domains, the molecule is stabilized by a central hydrophobic core. In
addition, a hydrophobic patch exists on the surface of the molecule
formed by the side chains of Val1151, Phe1152,
Val1153, and Leu1164.
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Fig. 4.
Sequence alignments of structurally related
biotin and lipoyl domains. The primary sequences of the biotin
domain of E. coli acetyl-CoA carboxylase (BDO)
and the lipoyl domains of A. vinelandii pyruvate
dehydrogenase (IYV), 2-oxoglutarate dehydrogenase
(GHJ), and B. stearothermophilus pyruvate
dehydrogenase (LAB) were aligned with the 104 C-terminal residues of yeast PC (YPC), as
described under "Experimental Procedures." Amino acid residues
conserved between the domains are colored white with
black background. Residues similar to those found
in the E. coli sequence at analogous positions are
shaded. The SCRs and nonconserved loop regions are indicated
above the alignment. The amino acids proposed to form
secondary structure in the yPC model are shown below the
alignment, as are the positions of residues present in yPC. The
biotinyl- and lipoyl-lysine are indicated by *.
Ala mutants were isolated,
Val1116, Val1148, and Val1166,
contribute to the hydrophobic core of the predicted structure and form
contacts with each other (Fig. 5). It is
reasonable to conclude that replacement of the larger Val side chain
with Ala distorts the packing arrangement of the hydrophobic core,
decreasing protein stability at the higher temperature. Our modeling
suggests that the two temperature-sensitive Ser
Pro mutations (Fig.
5) may introduce local conformational changes, which render the domain a less favorable substrate for BirA. The peptide backbone angles of
Ser1162 (
= +65°) are unfavourable for
introducing a Pro residue at this position (fixed
~
75°). Although a Pro substitution at Ser1141 (
=
94°) may be tolerated in this regard, this substitution may
abolish a potential interaction between the side chain of Ser1141 (strand 4) and the backbone carbonyl oxygen of
Gly1125 (strand 5), which are ~2 Å apart in the model.
Strands 4 and 5 define the hairpin loop containing Lys1135.
Distortion of the conformation of these strands, or the loss of
favorable interactions between them, is likely to alter the way the
loop is presented to the enzyme. Met1134 resides on this
loop adjacent to the critical lysine (Fig. 5); however, it does not
contribute to the hydrogen bonding in the hairpin, and so its
substitution is unlikely to destabilize this turn.
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Fig. 5.
Mutations affecting biotinylation of the
yeast PC biotin domain. A schematic stereo diagram of the
predicted structure of the yeast PC biotin domain showing the position
of the side chains of residues, which, when mutated, introduced
temperature sensitivity into the structure. The position of the biotin
accepting lysine at position 1135 and the adjacent Met1134
are shown. The figure was generated using Molscript, version 2.1.1 (46).
-helix (Fig. 4). The point mutations Ala1081
Pro and
Arg1083
Gly mapped to residues forming this
-helix.
Both substitutions introduced residues known to disrupt
-helices,
consistent with the notion that this region of the molecule may indeed
be structured.
Ile showed temperature sensitivity
in the in vivo biotinylation assays. This suggests that the
hydrophobic patch on the surface of yPC-104, containing
Phe1152, plays a role in the stability of the domain.
Furthermore, this mutant has a higher susceptibility to proteolysis,
supporting the involvement of Phe1152 in an interaction
stabilizing the domain. In the crystal structure of BCCP, the
corresponding residue (Leu139) interacts with another
hydrophobic residue (Ile78) found in the short
N-terminal tail outside of the structured domain (10). We
propose that the hydrophobic patch on the surface of yPC-104 may
interact with the N-terminal region of yPC-104, possibly
through a hydrophobic region on the predicted
-helix, and that the
change to Ile disrupts this interaction. We conclude that the observed
temperature sensitivity of the mutants we have isolated is consistent
with their proposed contribution within the structured biotinyl domain
of yPC-104.
Val and Met1134
Thr, were purified as unbiotinylated
domains (9) for further investigation. Molecular mass determination by
electrospray mass spectrometry confirmed the biotinylation state and
expected amino acid substitution of the purified proteins. The peptides
were 131 mass units less than the expected molecular mass (yPC-104: mass calculated 11,452.4; mass determined 11,322; Met1134
Val: mass calculated 11,420.3, mass determined 11,289;
Met1134
Thr: mass calculated 11,422.3, mass determined
11,292), suggesting the N-terminal Met (131.2) was excised
in vivo. The ability of these biotin domains to function as
substrates for BirA was assessed using steady state kinetics under
conditions that were optimal for biotinylation of BCCP-87 (Ref. 42;
Fig. 6, A and B).
BCCP-87 was the best substrate for the bacterial enzyme, whereas the
affinity of the wild-type yeast biotin domain for BirA was ~10-fold
lower (Table II). Similar
kcat values were observed for these two
substrates, suggesting that the deficiency in biotinylation was not due
to changes in the rate of catalysis. Biotinylation of the two yeast domain mutants could only be detected upon increasing the ligase concentration in the assay by 10-fold. Since we were unable to obtain
sufficiently high concentrations to obtain accurate kinetic constants
for the two mutant proteins, Km was estimated
from a double-reciprocal plot at low substrate concentration. This
indicated that both mutants had a Km for BirA at
least 10-fold higher than wild-type yPC-104 (Table II).
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Fig. 6.
In vitro biotinylation
assays. Purified apo-biotin domains were assayed with BirA
(A and B) or yBPL (C and D)
using the conditions described by Chapman-Smith et al. (42)
and Polyak et al. (9), respectively. The activity of the
enzymes was determined over the concentration range of apo-biotin
domains indicated on the graphs. The substrates analyzed were yPC-104
( ), Met1134
Val (
), Met1134
Thr
(
), and BCCP-87 (
). In the graph in A, 10-fold more
BirA was included in the reactions for the two yPC mutants. The lines
represent the nonlinear regression to the Michalis-Menten equation
using GraphPad Prism for MacIntosh (GraphPad Software Inc., San Diego,
CA).
Kinetic constants for the reaction of biotin domains with E. coli BirA
and yeast BPL
Val and
Met1134
Thr assayed with BirA and Met1134
Thr
assayed with yeast BPL were estimated from a double-reciprocal plot at
low substrate concentrations from the data shown in Fig. 6, as
discussed in the text. ND, not
determined.
Val mutant, which was a poor substrate for BirA, displayed only a
slightly lower affinity than wild-type yPC-104 (2-fold, p = 0.0002). As was observed with BirA,
Met1134
Thr was also a poor substrate for the yeast
enzyme displaying ~20-fold higher Km than
wild-type, estimated from a double reciprocal plot at low substrate
concentration. With yBPL as with BirA, similar rates of biotin transfer
(i.e. kcat) were observed for the
yeast and bacterial substrate proteins. Thus, the differences in
affinity (i.e.
kcat/Km) arise primarily
from differences in Km values.
Thr and
Met1134
Val--
As a second probe for the structural
integrity of the Met1134 mutants, the purified apo-proteins
were subjected to limited trypsin digestion. We have previously used
susceptibility to tryptic digestion to investigate the effect of point
mutations on the structure of a biotin domain (42). Digestion of
wild-type yPC-104 analyzed by SDS-PAGE showed an initial rapid cleavage
yielding a product with an apparent molecular mass of 11 kDa (Fig.
7). A second, slower cleavage generated a
stable product, which migrated at around 10 kDa. N-terminal
sequencing of this cleavage product revealed two scissile bonds between
Arg1083-Ser1084 and
Lys1086-Val1087, at or near the
C-terminal end of the proposed
-helix in the N-terminal extension. This product containing the
C-terminal structured biotin domain was relatively resistant
to further proteolysis, as it was the major species even after 5 h
of treatment (data not shown). The two purified mutant apo-biotin
domains were cleaved by trypsin at the same rate as the wild-type
protein and generated the same products (Fig. 7). These results
indicate that substitution of Met1134 with either Thr or
Val did not induce significant conformational changes to the structured
domain.
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Fig. 7.
Tryptic sensitivity of apo-yPC-104
mutants. Proteins were treated with trypsin using the conditions
described by Chapman-Smith et al. (42). Samples were taken
at various time intervals indicated above and analyzed by SDS-PAGE. The
panels show the digestion products of apo-yPC-104 (A),
apo-Met1134 Thr (B), and
apo-Met1134
Val (C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Val, was a poor substrate for both the
bacterial and yeast BPLs. In contrast, although the Met1134
Thr substitution was also a poor substrate for the bacterial BPL,
it was only a 2-fold poorer substrate for the yeast BPL than the
wild-type protein (Fig. 6, Table II). Our data show that there are
subtle differences in the active sites of BPLs from various species
which tolerate mutations to the flanking Met to varying degrees.
Furthermore, it has been known for some time that BPLs display cross
species reactivity (1, 3). Here we provide the first kinetic evidence
that BPLs biotinylate heterologous biotin domains with a lower
efficiency than they do their native substrates. Both the yeast and
bacterial BPLs displayed at least a 10-fold greater affinity for their
homologous substrates over heterologous biotin domains (Fig. 6, Table
II). Together these data indicate that, although BPLs catalyze the same
reaction, there are distinct differences between the two enzymes for
optimal substrate recognition.
-strands 1 and 2, and thus our positioning of
-strand 1 differs in register by two
residues. We also found no requirement for lengthening the loop between
-strands 7 and 8, and thus our alignment of
-strand 8 differs in
register by one residue position.
-helix with
three hydrophobic residues (Ile1078, Val1080,
and Ala1081), which may function as a cap, packing against
the surface of the biotin domain via the hydrophobic surface involving
residues Val1151, Phe1152, Val1153,
and Leu1164. We have previously shown that expression of a
peptide lacking the 23 N-terminal amino acid residues is a
6-fold less favorable substrate in in vivo biotinylation
assays than the 104 residue peptide (7). Without the
N-terminal extension, the domain was unstable during
purification and displayed a marked tendency to form high molecular
weight aggregates on gel filtration
chromatography.2 These
observations are consistent with our structural predictions and mutant
data, which suggest that the N-terminal extension plays a
role in stabilizing the overall domain structure, rendering the
molecule a better substrate for BirA.
2
and
3 (10, 11) that is not present in the structurally heterologous
lipoyl domains (Fig. 3 & 4). Interestingly, this thumb contacts the
biotin moiety in both the crystal (10) and solution (11) structures of
BCCP. The sequence of amino acids which form this thumb is absent in
most other biotin domains including P. shermanii TC (1, 3).
Furthermore, determination of the structure of the 1.3 S subunit of
P. shermanii TC has revealed that no thumb structure is
present in the domain (12) and the biotin moiety does not contact the
protein (43). In this regard, our predicted fold of yPC-104 more
closely resembles the structure of the 1.3 S subunit of TC. It is
possible that the absence or presence of the thumb on the biotin domain
contributes to the 10-fold reduction in affinity shown by the bacterial
and yeast BPL with a heterologous substrate.
-strands 1 and 2, which is close in space to
the lipoyl-lysine (Fig. 3 and 4; Refs. 14, 35, and 36). The residues
forming the surface loop are not required for in vivo
lipoylation, but instead are necessary for the reductive acetylation of
the prosthetic group (44). However, as biotinylation of the lipoyl
domain does not occur in E. coli in vivo (20), this surface
loop may also function to inhibit recognition by bacterial BPL. In the
BCCP domains, this middle finger loop is replaced by two residues
spanning the region (Fig. 4). The yPC-104 domain lacks both the thumb
(loop 2, Figs. 3 and 4) and the extended middle finger loop (loop 1, Figs. 3 and 4), further suggesting that structural differences govern
substrate recognition by yeast and bacterial BPLs.
-turn between
-strands
4 and 5 is essential in this process, as moving this residue by one place either side abolishes biotinylation (15). The kinetic data
presented here suggest that it is primarily the protein:protein interaction that is compromised by mutations to this scaffold rather
than the catalytic step, since Km values were affected to a greater extent than kcat values.
Although direct measurement of the binding interactions would address
this point more fully, it is technically difficult to do so. The form
of BPL that interacts with the apo-biotin domain is the
biotinyl-AMP-BPL complex, and once associated with an unbiotinylated
substrate, this complex turns over very rapidly to produce the
holo-biotin domain and free enzyme. To undertake such experiments
requires either a nonhydrolyzable analogue of biotinyl-AMP or a mutant biotin domain where the target Lys has been modified such that it
cannot accept biotin. We have produced a BCCP-87 mutant where the
target lysine has been substituted with Leu, but this appears to be a
poor inhibitor in studies using
BirA.3 It is possible that
BPL recognition absolutely requires the presence of a target Lys in a
structured biotin domain.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Denise Miller for assistance with protein sequencing, Dr. Yogi Hayasaka for performing electrospray mass spectroscopy, Dr. Dorothy Beckett (University of Maryland) for the kind gift of purified BirA, and Dr. Michelle Walker (University of Adelaide) for pMW4A. We also thank Prof. Murray Stewart (MRC Laboratory of Molecular Biology, Cambridge) for stimulating discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Australian Research Council Grant A09531996 (to J. C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia.
¶ Current address: Dept. of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia.
** To whom correspondence should be addressed: Dept. of Biochemistry, University of Adelaide, Adelaide, South Australia 5005, Australia. Tel.: 618-8303-5218; Fax: 618-8303-4348; E-mail: john.wallace@ adelaide.edu.au.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M003968200
2 A. Chapman-Smith and J. C. Wallace, unpublished data.
3 A. Chapman-Smith, S. Mortellaro, S. W. Polyak, J. Cronan, and J. C. Wallace, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BPL, biotin protein
ligase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
PCR, polymerase chain reaction;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis;
BCCP, biotin carboxyl carrier protein;
SCR, structurally conserved region;
TC, transcarboxylase complex.
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REFERENCES |
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