Department of Molecular, Cellular and
Developmental Biology, Yale University, New Haven, Connecticut
06520-8103,1 and Center for Metals in
Biocatalysis and Department of Biochemistry, Molecular Biology and
Biophysics, University of Minnesota Medical School, Minneapolis,
Minnesota 55455-03472
Protocatechuate 3,4-dioxygenase is a member of a family of
bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic
background, it is possible to select for Acinetobacter strains containing spontaneous mutations blocking expression of pcaH or -G, genes encoding the 
and 
subunits of protocatechuate 3,4-dioxygenase. The crystal structure of
the Acinetobacter oxygenase has been determined, and this
knowledge affords us the opportunity to understand how mutations alter
function in the enzyme. An earlier investigation had shown that a large
fraction of spontaneous mutations inactivating
Acinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant
selection was modified to isolate Acinetobacter strains in
which mutations within pcaH or -G cause a
heat-sensitive phenotype. These mutations affected residues distributed
throughout the linear amino acid sequences of PcaH and PcaG and
impaired the dioxygenase to various degrees. Four of 16 mutants had
insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large
structural changes can be tolerated. To further understand how protein
structure influences function, we isolated strains in which the
phenotypes of three different deletion mutations in pcaH or
-G were suppressed either by a spontaneous mutation or by a
PCR-generated random mutation introduced into the
Acinetobacter chromosome by natural transformation. The
latter procedure was also used to identify a single amino acid
substitution in PcaG that conferred activity towards catechol
sufficient for growth with benzoate in a strain in which catechol
1,2-dioxygenase was inactivated.
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INTRODUCTION |
Oxygenases are enzymes that split a
molecule of oxygen and introduce one or both of the oxygen atoms into
their substrates (18). The enzymes have long been the topic
of study because of their ability both to modify a wide variety of
stable substrates and to control highly reactive and potentially
dangerous oxygen species. These studies have demonstrated the important
roles of oxygenases in diverse organisms. In humans, for instance, the aromatic compound aspirin inhibits an oxygenase involved in the synthesis of steroids during inflammation (30). In plants,
an enzyme that appears to regulate programmed cell death has
significant sequence identity to bacterial oxygenases involved in
aromatic catabolism (15). The enzyme protocatechuate
3,4-dioxygenase is a member of a family of bacterial oxygenases
(18, 29) that use iron as a cofactor to cleave the aromatic
rings of their substrates between two adjacent hydroxyl groups, a key
step in mineralization of plant products (5). The crystal
structure of protocatechuate 3,4-dioxygenase from a strain of
Pseudomonas putida (originally classified as
Pseudomonas aeruginosa) has been determined (36, 37), and more recently, the structure of the enzyme from
Acinetobacter sp. strain ADP1 was solved (56,
57).
The product of protocatechuate oxygenase is carboxymuconate, and
Acinetobacter strains blocked in its metabolism fail to grow with any substrate when they are exposed to either protocatechuate or
its metabolic precursors. Thus, it is possible to select for strains in
which spontaneous secondary mutations block earlier steps in either
transport (6) or catabolism of aromatic compounds (Fig.
1). Strains derived from such selections
contained spontaneous mutations affecting residues important for
structure or function in various oxygenases:
p-hydroxybenzoate hydroxylase, encoded by pobA
(8, 21), protocatechuate 3,4-dioxygenase, encoded by
pcaH and pcaG (13), and vanillate
demethylase, encoded by vanA and vanB
(52). For genetic analysis of two regulatory proteins, PobR
(governing expression of pobA) and PcaU (governing
expression of pcaHG), PCR-generated mutations were
introduced into the Acinetobacter chromosome by natural
transformation, producing both loss-of-function and gain-of-function
mutations (26, 27).
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FIG. 1.
Positive selection of strains blocked in protocatechuate
catabolism. Strain ADP500 with the engineered  pcaBDK1
mutation cannot grow with succinate in the presence of protocatechuate
because of the toxic accumulation of
-carboxy-cis,cis-muconate. ADP500 derivatives
with spontaneous mutations in pcaH and -G, genes
encoding the two subunits of protocatechuate 3,4-dioxygenase, can
therefore be selected based on their resistance to protocatechuate.
Analogous to the conversion of protocatechuate to carboxymuconate by
protocatechuate 3,4-dioxygenase (PcaHG), catechol 1,2-dioxygenase
(CatA) converts catechol to muconate in the benzoate branch of the
-ketoadipate pathway.
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The present investigation is a genetic analysis of
Acinetobacter protocatechuate 3,4-dioxygenase complementing
the recently determined crystal structure of the enzyme (56,
57). A refinement in the positive-selection protocol enabled
rapid identification of strains with a heat-sensitive mutation in
pcaH or -G, and subsequently, strains with
spontaneous or PCR-generated second-site suppressors of some of these
mutations were selected. Transformation-facilitated PCR mutagenesis was
also used to select one strain in which a single amino acid
substitution in PcaG allowed protocatechuate 3,4-dioxygenase to
functionally replace catechol 1,2-dioxygenase (Fig. 1).
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MATERIALS AND METHODS |
Strains and culture conditions.
Acinetobacter sp.
strain ADP1, originally designated Acinetobacter
calcoaceticus BD413 (24), was routinely grown with 10 mM succinate in a mineral medium (13). Unless otherwise
indicated, cells were grown at 37°C and the mineral medium was
supplemented with 5 mM p-hydroxybenzoate, 5 mM quinate, 3 mM
protocatechuate, or 2.5 mM benzoate. Because of the instability of
protocatechuate, we used only fresh plates with this carbon source,
made with stock solutions (pH 7.0) that had been stored frozen until
use. Escherichia coli strains (50), namely,
DH5 (purchased as competent cells from Gibco BRL) and TG2 (gift of
M. Biggin and T. Williams), were grown with Luria-Bertani medium.
Ampicillin was added at 100 µg/ml to select for E. coli
cells with plasmids derived from pUC18 or pUC19 (59), and
tetracycline was added at 12.5 µg/ml to select for E. coli
or Acinetobacter cells with plasmids derived from pRK415
(25).
DNA manipulations.
Crude cell lysates of
Acinetobacter strains for use in transformation reactions
were prepared by incubation of pelleted cells (from a 5-ml culture) in
0.5 ml of saline-citrate buffer with 0.05% sodium dodecyl sulfate at
60°C for 1 h (24). Plasmids were isolated from 5-ml
E. coli cultures with a Wizard miniprep kit (Promega) and
from 5-ml Acinetobacter cultures by the alkaline lysis
miniprep procedure (50) with two extraction steps, the first
with phenol-chloroform and the second with chloroform. To isolate
template DNA for PCR, pelleted Acinetobacter cells from a
5-ml culture were washed and treated with InstaGene Matrix as recommended by the supplier (Bio-Rad); 5 µl of the resulting solution was then used in 50-µl PCR mixtures including 0.5 U of Taq
polymerase (Boehringer Mannheim), 10 pmol of each primer, and 10 nmol
of each deoxynucleoside triphosphate. Standard PCR conditions were used: 30 cycles of denaturing at 94°C for 45 s, annealing at
56°C for 45 s, and elongation at 72°C for 1 min 30 s. PCR
primers were synthesized by the Keck Biotechnology Resource Lab (Yale University).
Selection and characterization of strains with a spontaneous
heat-sensitive mutation in pcaH or -G.
Strain
ADP500 (13, 20) contains the engineered
catD101::Kmr and
pcaBDK1 mutations. Single colonies of succinate-grown
ADP500 were transferred to patches on freshly prepared plates with 10 mM succinate and 3 mM protocatechuate. Cells with spontaneous secondary
mutations blocking protocatechuate catabolism do not accumulate toxic
levels of -carboxy cis,cis-muconate and are able to grow (Fig. 1). To prevent analysis of siblings, only one mutant
derivative was picked per single colony of ADP500.
After incubation for 2 days at 37°C, colonies of ADP500 derivatives
with secondary mutations were transferred to each of three plates: one
plate contained the original selective medium and two plates contained
10 mM succinate and 5 mM p-hydroxybenzoate. The latter two
plates were incubated for 2 days, one at 37°C and one at 22°C.
Strains with a conditional resistance to p-hydroxybenzoate (with significantly better growth at 37°C than at 22°C) were picked from the plate with protocatechuate and purified by two further rounds
of growth on plates in the presence of protocatechuate. The
pcaBDK1 deletion was restored to wild type by
transformation with plasmid pZR3 (13, 20) under nonselective
conditions, followed by selection for growth with benzoate. Expression
of PcaD in recombinants is sufficient for growth with benzoate and complements the engineered
catD101::Kmr mutation inactivating
the isofunctional enzyme in the benzoate branch of the -ketoadipate
pathway (13, 20).
Each of the resulting strains (ADP1102 to -1117) contained a
spontaneous mutation blocking protocatechuate catabolism. Single colonies of succinate-grown mutant cells were then tested for growth,
at 37 and 22°C, on plates with p-hydroxybenzoate as the sole carbon and energy source. The heat-sensitive mutation in either
pcaH or -G was localized by marker rescue as
described previously (13) except that quinate was used as
the selective growth substrate (Fig. 1). DNA transformations for marker
rescue or linkage experiments were performed by growing recipient
Acinetobacter strains with 10 mM succinate overnight in 5-ml
cultures. After addition of 10 µl of 1 M succinate, these cultures
were then incubated (to induce competence) at 37°C for 30 min in a
gyratory shaker before being spread onto selective plates onto which
donor DNA was then spotted.
Recovery of Acinetobacter chromosomal DNA by gap
repair.
DNA fragments containing mutant pcaH and
-G genes were recovered from the chromosome by gap repair
with plasmid pZR1007 as previously described (2, 16). To
reduce the chance that the recovered DNA would reintegrate into the
chromosome, the duration of steps involving Acinetobacter
cells was minimized. The 2.4-kb HindIII restriction
fragment containing pcaK'CHGquiB' (13, 19) from
each pZR1007 derivative was purified by preparative gel electrophoresis and ligated with HindIII-digested pUC18, and the
resulting plasmids were amplified in E. coli DH5. Cells
with plasmids in which the insert was oriented so as to be expressed
from the vector promoter formed distinctively small colonies on
Luria-Bertani medium plates with ampicillin. Minipreps of such cells,
however, gave low plasmid yields, so the plasmids were transferred to
E. coli TG2 cells (in which lacIq
decreases the potentially toxic uninduced expression of the cloned genes).
Selection and characterization of a strain in which
protocatechuate 3,4-dioxygenase functionally replaced catechol
1,2-dioxygenase.
To select strains in which a gain-of-function
mutation in protocatechuate 3,4-dioxygenase conferred activity towards
catechol, pcaH and -G were first inserted into
the gene for catechol 1,2-dioxygenase (catA) so that their
expression would be regulated by benzoate (4). Plasmid
pIB1344 (35), which contains catA, was cut with SalI and recircularized, generating pZR7559 with a unique
SphI site within catA. A DNA fragment containing
pcaC'HG (in which the 
element from pHP45
[44] had been inserted into the BamHI site
in pcaH) was amplified with Pfu polymerase
(Stratagene) with plasmid pZR200 (gift of M. Stein) as the template
and primers HG1 and HG6 (see below). The PCR DNA and
SphI-digested pZR7559 were blunted with the DNA polymerase
Klenow fragment and ligated together, and transformants of E. coli DH5 that were resistant to streptomycin, spectinomycin,
and ampicillin and contained plasmids in which pcaHG and the
disrupted catA were in the same orientation were selected.
The insert from one such plasmid was introduced into the chromosome of
Acinetobacter strain ISA25 (catBCIJF) by
selecting for transformants in which inactivation of catA
prevented the toxic accumulation of muconate generated during growth
with succinate in the presence of benzoate (12, 58). PCR
amplification across catA in one Smr
Spcr Amps transformant with primers catA1 and
catA2 (see below) and an extension time of 1 min 30 s generated a
fragment of 3.4 kb, consistent with the absence of the 2-kb element
within pcaH. This PCR DNA was used, as before, to generate
an ISA25 transformant strain resistant to benzoate, and the
catBCIJF deletion in one such strain was corrected by
transformation with pPAN4 (35, 53) and selection for growth
with muconate, generating ADP7559. Slow growth with muconate of ADP7559
suggested the existence of a spontaneous mutation affecting muconate
transport (58), as has been observed for ISA25 derivatives
in two separate studies (3, 58).
PCR mutagenesis of ADP7559 generated ADP7615. The pcaG7615
gain-of-function mutation in the latter strain was recovered from the
chromosome by PCR amplification with the primers catAK
(5'-GGGGGTACCCTGATTCTACATGGCACG-3') and catAR
(5'-GGGGGAATTCATCGGTAATAATACTACGGCG-3') whose 5'
ends include KpnI and EcoRI restriction sites
(underlined), respectively. After digestion with KpnI and
EcoRI, this PCR fragment was ligated with KpnI-
and EcoRI-digested pUC19, generating pZR7615 in which pcaH and -G are expressed from the plasmid
promoter. pZR7615 was transferred from E. coli DH5 cells
to TG2 cells, and the sequences of the cloned genes containing
pcaG7615 were reconfirmed.
Transformation-facilitated PCR mutagenesis.
For mutagenesis
(26, 27), standard PCR was performed as described above
except that the number of cycles was increased to 35. To generate DNA
with suppressors of a heat-sensitive pcaH or -G
deletion, primers HG1 (5'-CTACATTGTTCACTTTATGCAGGC-3') and HG6 (5'-GATATACGGCCCGTTCCATAGTC-3') were used, amplifying a
1.5-kb PCR fragment. To mutagenize the transposed pcaH and
-G genes in ADP7559, primers catA1
(5'-GGTATAGAAACGACTATCG-3') and catA2
(5'-CAAGTGTATGTCGTAACGC-3') were used, amplifying a 3.4-kb
fragment. PCR-amplified fragments, generated with template DNA from the
planned recipient strain, were used directly in transformation reaction
mixtures as follows. Two hundred microliters of a 5-ml culture of the
recipient strain, grown overnight with 10 mM succinate, was transferred
to a fresh 5-ml culture, and after growth in a gyratory shaker for
2 h (to induce competence), 500 µl of the culture was
transferred to Falcon 15-ml polypropylene tubes together with 20 µl
of the PCR mixture. After overnight shaking incubation, transformation
reaction mixtures were spread onto selective plates.
DNA sequence analysis.
To isolate template DNA for
sequencing, PCR mixtures prepared under standard conditions were
purified with 8 µl of GeneClean Glassmilk according to the
recommendations of the supplier (Bio 101, Inc.) and resuspended in 25 µl of water, and 8 µl was used for ABI PRISM Dye Terminator Cycle
Sequencing with AmpliTaq DNA polymerase FS (Perkin-Elmer). Cycle
sequence reaction mixtures were processed as previously described
(26).
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RESULTS |
Characterization of strains with a spontaneous heat-sensitive
mutation in pcaH or -G.
By positive selection
for derivatives of Acinetobacter strain ADP500 that have a
spontaneous mutation blocking the -ketoadipate pathway, 94 strains
(13) that had a mutation affecting protocatechuate 3,4-dioxygenase, the enzyme which cleaves the protocatechuate aromatic
ring generating carboxymuconate (Fig. 1), were characterized. Only 11 of the 94 strains, however, had a missense mutation in pcaH
or -G, a gene encoding the or subunit of the
dioxygenase, respectively. Nearly one-quarter of the mutants had a
deletion extending into DNA outside of these two genes (13).
Six of the missense mutations caused a heat-sensitive phenotype,
blocking growth with protocatechuate at 37°C but not at 22°C
(13). It therefore seemed feasible to use this phenotype as
a way to identify, early in the selection protocol, those ADP500
derivatives most likely to have a spontaneous missense mutation in
pcaH or -G. This identification process would
allow efforts to be focused on strains which could contribute to
understanding how structure influences function in the dioxygenase
enzyme and eliminate time spent identifying mutants with large
deletions, frameshifts, or nonsense mutations.
To identify heat-sensitive pcaH or -G mutants in
this study, ADP500 derivatives still containing the
pcaBDK1 deletion and able to grow in the presence of
protocatechuate at 37°C were tested for resistance to
p-hydroxybenzoate during growth with succinate at both 37 and 22°C (Fig. 1). Screening was done with
p-hydroxybenzoate because cells were found to grow poorly
upon successive transfers onto plates containing protocatechuate, a
compound that has inherent toxicity (41). Consistently,
approximately 5% of the spontaneous mutants analyzed were resistant to
p-hydroxybenzoate at 37°C but not at 22°C. After
correction of the pcaBDK1 deletion in 17 such strains,
the expected heat-sensitive phenotype was revealed: growth with
p-hydroxybenzoate was completely blocked at 37°C but
allowed at 22°C. The spontaneous mutation in each of these 17 strains, with one exception, was mapped by marker rescue to
pcaH and pcaG and sequenced. The one unique
strain had a point mutation in the gene for PcaU, the transcriptional
activator governing pca operon expression, and its
characterization is part of a separate study (51).
The pcaH and -G mutations and their associated
amino acid changes in the heat-sensitive mutants isolated in this study
or previously (13) are listed in Table
1. Figure 2
shows the positions of the mutations in the primary sequence of
Acinetobacter PcaH and -G. The pcaG13 and
pcaH1112 point mutations were each found in two
independently isolated strains, and the pcaG13 and
pcaG1114 mutations changed the same nucleotide but caused
different amino acid substitutions (Table 1). Most of the remaining
mutations affected residues distributed throughout the linear amino
acid sequence of PcaH and -G (Fig. 2). Although all the mutants grew with p-hydroxybenzoate at 22°C but not at 37°C,
protocatechuate 3,4-dioxygenase appeared to be impaired to various
degrees: the most severely affected mutants (ADP6116 and ADP1116) grew
with p-hydroxybenzoate extremely slowly even at 22°C.
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FIG. 2.
Spontaneous and PCR-generated mutations in
protocatechuate 3,4-dioxygenase from Acinetobacter sp.
strain ADP1. Protocatechuate 3,4-dioxygenase is an oligomer
(29) of heterodimers composed of PcaG (the subunit) and
PcaH (the subunit). The two subunits have significant amino acid
identity, and it has been suggested that they are derived from
duplication and divergence of the gene for the homodimer subunit of the
ancestral enzyme (36, 37). Acinetobacter
protocatechuate 3,4-dioxygenase subunits (GenBank accession no. L05770)
are presented so that homologous amino acid sequences occupy
corresponding positions in PcaG (top) and PcaH (bottom). In order to
achieve this alignment, gaps indicated by dashes were introduced into
the sequences. With the exception of pcaG7615, G425A,
nucleotide sequence changes causing the amino acid alterations shown
here are presented in Tables 1 and 2. Alleles with designations smaller
than 1000 were identified in a previous investigation (13).
To facilitate comparison with structural data from previous studies
(37, 56), the same numbering system has been used for the
primary structure with PcaG amino acids numbered 1 to 200 and PcaH
amino acids numbered 300 to 540 (the five Acinetobacter PcaG
residues that align with a gap between residue 88 and 89 in P. putida PcaG are designated 88a to -e). Amino acid substitutions
are indicated by arrows pointing to the mutant amino acid. Duplicated
residues are underlined, and the mutant repetitions are shown above the
primary sequence. Shaded boxes superimposed on the primary sequence
indicate residues missing in deletion mutations. Amino acids in shaded
boxes separated by arrows from the primary sequence indicate suppressor
mutations, and the borders of the boxes (solid, dotted, or not
bordered) match those of the shaded boxes indicating the mutations that
are suppressed. The double-dotted border indicates a mutation that
confers the ability to grow at 37°C to a strain containing both
pcaG1102 and pcaG7554. The R133H substitution
caused by pcaG7615 and conferring activity towards catechol
is indicated by a box enclosing the structure of the compound. The
glycyl residue at position 60 in PcaG appears to be a natural variant
and is found in all the mutants described in this study (including
ADP7615), whereas the wild-type strain ADP1 has serine (indicated in
parentheses in the figure) at the corresponding position.
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The majority of strains had a missense mutation, but unexpectedly,
protocatechuate 3,4-dioxygenase was more severely altered in four newly
isolated strains. Deletions in pcaH1116 and
pcaG1102 caused deletions of 1 and 10 amino acid residues,
respectively, and mutations in pcaH1111 and
pcaG1103 caused insertions of 1 and 2 amino acids,
respectively (Table 1; Fig. 2). Both deletions may have arisen by
recombination between directly repeated DNA sequences, resulting in
loss of one of the repeats, TAT in the pcaH1116 strain
and GATAC in the pcaG1102 strain. The 3-bp
pcaH1116 deletion appears to lie within a local region of
DNA instability, given its proximity to the previously described
overlapping null mutations pcaH21 and pcaH13,
duplications of 3 and 7 bp, respectively, (13). The cause at
the DNA level of the duplication of TTC in pcaH1111 and
AAATGC in pcaG1103 is likely to have been
mediated by slippage at the tandem DNA repeats immediately flanking
each of these mutations, GGTGGT and GAAGCAGAAGCA,
respectively. As expected, phenotypic revertants were readily
obtained from strains carrying either of these duplications.
Suppression by intragenic and extragenic point mutations.
Strains in which a pcaH or -G deletion did not
cause a null phenotype were particularly attractive candidates to use
to identify second-site suppressor mutations since the encoded enzyme
was still partly functional and a background of direct reversion would not be a concern. Suppression was first tried with a strain from a
previous study, ADP6338, containing the pcaH7 leaky
4-amino-acid deletion which causes slow growth with
p-hydroxybenzoate at both 37 and 22°C (13)
(Fig. 2). Liquid cultures of ADP6338 grown with 10 mM succinate and 5 mM p-hydroxybenzoate were serially transferred, and after
the third transfer, one culture failed to develop the purple color
indicative of protocatechuate accumulation due to deletion of
pcaH7. Partial suppression of the pcaH7 leaky phenotype in a purified isolate from this culture was confirmed by
comparing the growth on a plate with p-hydroxybenzoate of
the derived strain, designated ADP7552, with that of ADP6338.
In addition, occasionally a liquid culture shaking at 37°C would not
grow when it was provided with 5 mM p-hydroxybenzoate as the
carbon source and inoculated with a single colony of succinate-grown ADP6338. In one case, the culture suddenly became turbid after 18 days
of incubation. A purified isolate, designated ADP7553, grew slightly
faster than the parental strain (but not as well as ADP7552) on a plate
with p-hydroxybenzoate at 37°C. The cause of the varied
behavior of ADP6338 is unknown but may reflect different physiological
states conferring upon cells a range of sensitivities to the sudden
intracellular accumulation of protocatechuate generated during growth
with p-hydroxybenzoate.
Evidence that a mutation genetically linked to the pcaH7
deletion was the cause of suppression in both ADP6338 derivatives came
from results of a DNA transformation experiment using a recipient strain with the 440-bp pcaCH1 deletion (13)
encompassing the pcaH7 deletion locus: recombinants that
grew at 37°C with p-hydroxybenzoate like ADP7552 or
ADP7553 were readily obtained with donor DNA from the corresponding
strain either in crude cell lysate or as a 2.4-kb HindIII restriction fragment containing
pcaCHG recovered from the chromosome by gap repair (2,
16). Confirming this linkage, sequencing of pcaH and
-G in each strain revealed that protocatechuate 3,4-dioxygenase contained, in addition to the deletion of PcaH residues
319 to 322 due to the pcaH7 deletion, a G13S change in PcaG
in ADP7552 and a P317L change in PcaH in ADP7553 (Fig. 2; Table
2).
During this investigation but as part of a separate study, PCR
mutagenesis was combined with natural transformation to obtain loss-of-function as well as gain-of-function mutations in the PobR
regulatory protein (26, 27). To show in that study that the
technique was broadly applicable for genetic analysis of chromosomal genes in Acinetobacter, we identified PCR-generated
mutations that suppressed the heat-sensitive pcaG1102
deletion, causing loss of PcaG amino acid residues 76 to 85 (26) (Fig. 2). Each suppressor mutation caused an amino acid
substitution near the deletion in the PcaG linear amino acid sequence
and restored the ability to grow with p-hydroxybenzoate at
30°C but not at 37°C (26) (Fig. 2; Table 2). However,
during a later experiment using cells with the pcaG7554
suppressor, a single colony that could grow with
p-hydroxybenzoate at 37°C appeared, suggesting that
further spontaneous suppression of the heat-sensitive phenotype had
been selected. Subsequent sequencing of pcaH and
-G in this strain revealed an additional point mutation
causing a D74N substitution, again near the original mutation in the
PcaG primary sequence (Fig. 2; Table 2).
Of the heat-sensitive pcaH or -G mutations in
Table 1, deletion of pcaH1116 caused one of the strongest
phenotypes: strains with this mutation grew with
p-hydroxybenzoate extremely slowly even at 22°C.
Nevertheless, PCR mutagenesis also successfully suppressed deletion of
pcaH1116, generating a strain in which an L465V substitution
in PcaH in combination with the deletion of Y437 caused by the
pcaH1116 deletion restored growth with
p-hydroxybenzoate even at 37°C (Fig. 2; Table 2). PCR
amplification across both mutations in the suppressed strain created
DNA that gave rise to over 1,000 times the number of transformants in
the initial selection, consistent with the sequenced second-site
mutation being the cause of suppression.
An alternate form of suppression.
PCR-generated suppressor
deletions of pcaG1102 were found in colonies that appeared
on a plate with p-hydroxybenzoate after overnight incubation
at 30°C. At this time there was no growth on control plates of
ADP1102 cells that had not been transformed with PCR DNA. However,
after several days' further incubation, the control plates usually
contained several colonies with exceptional properties. These strains
maintained the ability to grow at 30°C with
p-hydroxybenzoate even after the selective pressure was
relieved during successive rounds of growth with succinate, suggesting that the strains had acquired a heritable suppressor mutation. Yet, DNA
sequencing of pcaH and -G in two such strains
revealed only the original pcaG1102 mutation and DNA in
crude cell lysates of these strains could not transform the new
phenotype into the parental strain.
It has been noted that during suppression analysis of bacteria, often
the most readily isolated cells are those in which amplification on the
chromosome of the partly dysfunctional gene generates sufficient mutant
protein activity to allow growth under the selective conditions (49). Such a phenomenon may account for the properties of
the strains in which the pcaG1102 deletion appeared to have
been spontaneously suppressed. DNA amplification has also been shown to
be an adaptive response of wild-type bacterial cells (33,
46-49). In Acinetobacter, the ability to coamplify
(49) all the genes necessary for catabolism of
protocatechuate, quinate, and p-hydroxybenzoate to citric
acid cycle intermediates is a selective benefit that may have acted continuously over evolutionary time to favor the current supraoperonic clustering (2, 14, 22) of these genes.
A single amino acid substitution in PcaG (R133H) confers catechol
1,2-dioxygenase activity.
Isolation of strains with a
PCR-generated suppressor mutation as described above suggested that the
technique might be a powerful method of isolating gain-of-function
pcaH or -G mutations. In the benzoate branch of
the -ketoadipate pathway, catechol 1,2-dioxygenase occupies a
position equivalent to that of protocatechuate 3,4-dioxygenase, and the
respective substrates of these two enzymes differ by only a carboxyl
group (Fig. 1). Comparison of the amino acid sequences of the two
oxygenases indicates common ancestry (19, 34). An intriguing
question, therefore, is to what extent the two enzymes have specialized
to perform their respective tasks since their divergence from a common
ancestor (55).
To address this problem, we constructed strain ADP7559, in which a copy
of pcaH and -G was inserted near the middle of
catA (34), the gene encoding the subunits of the
catechol 1,2-dioxygenase homodimer (42). This insertion
prevented growth with benzoate but presumably allowed benzoate to
induce expression of the transposed genes (4). PCR
amplification across pcaH and -G in this strain generated DNA which gave rise to transformants of ADP7559 that could
grow with benzoate, although the brown staining produced during growth
of the transformants indicated some accumulation of catechol. No
background of spontaneous mutants was detected. Sequencing of the
transposed pcaH and -G in 13 transformants, each
independently generated by different PCRs, revealed in every case the
same G425A point mutation (pcaG7615) causing an R133H substitution in PcaG (Fig. 2). The pcaH and -G
genes from within catA in one of the transformants (ADP7615)
were cloned by PCR into plasmid pZR7615, and after confirmation of the
DNA sequence, transformation with this DNA was shown to confer the
ability to grow with benzoate upon the parental strain.
| |
DISCUSSION |
Genetic analysis of protocatechuate 3,4-dioxygenase structure and
function.
In a previous investigation using a parental strain with
the pcaBDK1 deletion, derivatives in which a conditional
mutation in pcaH or -G provided resistance to the
toxic intracellular accumulation of carboxymuconate produced from
protocatechuate in the medium were isolated (13) (Fig. 1).
These mutations were identified after pcaBDK DNA was
restored to wild type. In contrast, this study describes a protocol for
the identification of strains with a conditional pcaH or
-G mutation before any genetic manipulation, thereby
facilitating the generation of a large collection of such strains
useful for an analysis of the effects on protocatechuate 3,4-dioxygenase function of relatively subtle alterations in the structure of the protein.
The pcaH and -G mutations isolated in this study
and previously (13) have a range of heat sensitivity
phenotypes. Unexpectedly, one-quarter of the mutants produced in this
study had small deletions or insertions in pcaH or
-G. The largest deletion (pcaG1102) removed 10 amino acids from a surface loop in the subunit that contacts the
subunit of the enzyme (37, 57). The sequences of this
loop are highly varied among the organisms for which the sequences are
known, with the Acinetobacter strain ADP1 and
Rhodococcus opacus 1CP enzymes having five additional
residues and the Burkholderia cepacia and Pseudomonas
marginata enzymes having two additional residues relative to the
number of residues in the corresponding loop in the P. putida enzyme (10, 11, 19, 43, 60). This finding
illustrates how the positive-selection protocol used here reveals not
only general structural determinants of protein stability (1, 31,
32, 40) but also regions of the protein where large alterations
can be tolerated.
Thermodynamic experiments have shown that the free energy of folding of
proteins is relatively small, approximately 5 to 15 kcal/mol
(45). This small value favoring protein folding is due to a
delicate balance between two large numbers, the free energy of the
noncovalent interactions in the folded state and that in the unfolded
state. Interactions between hydrophobic residues are the principal
thermodynamic force in the stabilization of the folded state, while
solvation energies are the principal destabilizing force. Computational
analyses of models of protein folding (7) have shown that
mutations can lead to large changes in the denaturation temperature of
a protein with little change to the overall structure of the protein.
Thus, the protein can be active at reduced temperatures but inactive at
higher temperatures.
In a method similar to the plating selection used to obtain mutants of
pcaH and -G, bacteriophage T4 lysozyme mutants in
which the enzyme was phenotypically inactive at 42°C were obtained
(17, 23). When purified, these enzymes were found to have
reductions in thermal stability that could be rationalized upon
examination of the structure. The recently determined crystal structure
of protocatechuate 3,4-dioxygenase from Acinetobacter sp.
strain ADP1 (57) similarly facilitates analysis of the
mutations in this study for their possible effects. Figure
3 shows the positions of all
temperature-sensitive, deletion, and suppressor mutations superimposed
on a drawing of the C trace of the Acinetobacter enzyme.
Table 3 describes the structural
consequences of the mutations. Typically, the mutations create van der
Waals clashes, cavities in hydrophobic regions, or isolated buried
charges destabilizing the native structure of an individual subunit, of
an intersubunit interface, or of the interface between molecules in the
dodecameric aggregate. For three of the mutations, residues in the
active site are involved so that the reduction in activity may not
arise from a lack of stability. T12I abolishes hydrogen bonds which stabilize the main chain around Pro15. Since the side chain of Pro15
forms part of the narrow waist of the substrate binding site, changes
in its position may allow protocatechuate to bind to the iron in less
productive orientations. W400S eliminates a residue that stacks against
His462, an iron ligand. The substitution also removes the N
that may
form a hydrogen bond with molecular oxygen during the reaction cycle.
P458L may move the end of Arg457, which has been proposed to stabilize
the development of a negative charge on C-4 of protocatechuate,
allowing an electrophilic attack by molecular oxygen (38).
View larger version (51K):
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|
FIG. 3.
Stereoview C trace of protocatechuate 3,4-dioxygenase
from Acinetobacter sp. strain ADP1 showing the positions of
spontaneous and PCR-generated mutations. Each protomer of
protocatechuate 3,4-dioxygenase is composed of an subunit (dashed
line), a subunit (solid line) and one nonheme iron (black sphere).
Each mutation is labeled with a sphere and a letter which corresponds
to a letter in Table 3. Point mutations and insertions are in white
spheres. These mutations are scattered throughout the structure but
usually lead to disruption of interfaces between secondary or tertiary
structural elements. The deletion mutations X, Y, and Z are depicted in
green, blue, and red spheres, respectively. Rescue mutations are shown
in lighter shades of the corresponding color.
|
|
The three pcaH or -G deletion mutations for which
second-site suppressors were identified in this study (Fig. 2 and 3;
Table 2) appear to affect protocatechuate 3,4-dioxygenase in distinct ways: the pcaH7 deletion causes the loss of 4 amino acid
residues in a loop containing Tyr324, which forms a hydrogen bond with the substrate in the native enzyme (38, 57); the
pcaH1116 deletion causes the loss of one residue at the
amino terminus of a strand in the flattened barrel of the subunit; and deletion of pcaG1102 causes the loss of 10 residues in a loop that forms a portion of the interface between the
and subunits (37, 57). The fact that spontaneous or
PCR-generated suppressor mutations were readily identified for each of
these deletions, with the pcaH7 deletion being suppressed by
either an intragenic or an extragenic mutation (Fig. 2; Table 2),
demonstrates the usefulness of combining positive selection and natural
transformation for genetic analysis of protocatechuate 3,4-dioxygenase
in Acinetobacter strain ADP1. Furthermore, it was
discovered, unfortunately in a separate investigation after this study,
that the combination of a mutation inactivating the regulatory gene
pcaU with the leaky pcaH7 structural gene
mutation completely blocked growth with p-hydroxybenzoate or
protocatechuate although either mutation alone did not (27).
This double mutant was used to identify PCR-generated gain-of-function
mutations in pobR which could confer PcaU activity
(27). However, the same strategy should also greatly facilitate the identification of PCR-generated mutations in
pcaH or -G that suppress the pcaH7
deletion or other leaky mutations that subtly alter the enzyme, since
strains with such suppressors would appear against a background of no
growth. Further biochemical and structural analysis of these suppressor
mutations might shed light on the long slow process by which proteins
evolve over time by accumulating beneficial mutations in combinations
of two or more.
It has long been noted that the two parallel branches of the
-ketoadipate pathway (Fig. 1), the p-hydroxybenzoate
branch encoded by genes in the pca-qui-pob supraoperonic
cluster (14, 22) and the benzoate branch encoded by the
ben-cat supraoperonic cluster (14, 22), represent
an attractive system for studying how the demands for a set of chemical
reactions in a single cell line were met twice over evolutionary time
to create each of the two branches of the pathway (55). At
the two extremes of a continuum, the coenzyme A transferases encoded by
pcaIJ and catIJ are over 99% identical
(28) whereas the lactonizing enzymes encoded by pcaB and catB, although they act on closely
related substrates, come from distinct protein families and do not have
significant sequence identity (28). Between these extremes,
the two dioxygenase subunits encoded by pcaH and
-G have 26 to 36% amino acid identity over 127 to 213 aligned residues with the homodimer subunit encoded by catA,
suggesting that the two enzymes have a common ancestor. Nevertheless,
it was unexpected that it would take one and only one amino acid
substitution in protocatechuate 3,4-dioxygenase to confer activity
towards catechol sufficient to allow growth with benzoate in a strain
in which catA was inactivated.
The ease with which directed evolution may generate a protocatechuate
3,4-dioxygenase capable of functionally replacing catechol 1,2-dioxygenase is intriguing given that there appears to be one trait
which consistently distinguishes the two bacterial dioxygenases: the
oligomeric complexity of the enzymes. Protocatechuate 3,4-dioxygenase forms oligomers composed of different numbers of -subunit
heterodimers in different bacteria (29): 12 in
Acinetobacter (56) and P. putida
(37) and 6 in Brevibacterium fuscum
(9). Catechol 1,2-dioxygenases are not known to oligomerize
beyond homo- and heterodimers (18). Oligomerization has been
suggested to enhance thermal stability (37), perhaps
indicating a trade-off between stability and catalytic efficiency
(54). The active site in Acinetobacter
protocatechuate 3,4-dioxygenase is at the interface near a
neighboring protomer related by the local threefold-symmetry axis
(57). Near the corresponding symmetry axis in the P. putida enzyme there is a second site where the substrate and
inhibitors can bind (38, 39), being connected to the active
site by a solvent-filled channel. These observations fueled speculation that the oligomeric state of these intradiol dioxygenases plays an
important role in function. The finding of the R133H gain-of-function mutation shows that the oligomeric state may play only a small role in
substrate specificity and that this can be overcome by a single mutation.
The opening to the active site of Acinetobacter
protocatechuate 3,4-dioxygenase is at the bottom of a 15-Å-deep cavity
surrounded by a preponderance of basic residues. Calculation of the
electrostatic potential around P. putida protocatechuate
3,4-dioxygenase shows that a large sphere of positive electrostatic
potential originates around the iron and extends all the way to the
outer edge of the active-site cavity (39). It has been
proposed that the enzyme uses this excess positive potential to attract
negatively charged substrates like protocatechuate into the active
site. Arg133 is one of the positively charged residues in the
midsection of the active-site cavity. It makes an intrasubunit salt
link to Asp65 and an interprotomer salt link to Glu162. Arg133 and
other positively charged residues near the entrance to the active-site
cavity are not conserved in the catechol 1,2-dioxygenases. Modeling of
the R133H mutation indicates that a histidine at this position would not be able to form a direct interaction with Glu162 of the neighboring protomer. Since both residues are exposed, the free-energy penalty of
disrupting this interaction should be on the order of 1 kcal/mol or
less. Choosing a common rotamer of the histidine would place the ring
of the side chain within hydrogen-bonding distance of Tyr324 and
Thr326. In the structure of the oxygenase with bound protocatechuate
(38, 57), Tyr324 interacts with the carboxyl of the
substrate. If the histidine rotated into this area it might interact
directly with catechol, facilitating more productive modes of binding
during catalysis with catechol.
Yet to be determined is the extent to which the R133H mutation deprives
protocatechuate 3,4-dioxygenase of its capacity to support growth with
protocatechuate in the wild-type genetic and physiological context of
pcaH and -G. In short, did the gain-of-function mutation make the enzyme a generalist, capable of acting effectively upon both catechol and protocatechuate, or a shifted specialist, with
effective activity changed from protocatechuate to catechol?
This research was supported by grants DAAG55-98-1-0232 from the
Army Research Office and MCB-9603980 from the National Science Foundation to L.N.O. and by grants from the National Institutes of
Health (GM-46436) and from the Minnesota Supercomputer Institute to
D.H.O. M.W.V. acknowledges an NIH predoctoral training grant (GM-08277).
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Abstract
Introduction
Publication 21 from the Biological Transformation Center in the
Yale Biospherics Institute.
Present address: Department of Genetics, University of Washington,
Seattle, WA 98195-7360.
