From the Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02115, the
¶ Departament de Ciències Mèdiques Bàsiques,
Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Spain, and
the
Departament de Bioquímica, Facultat de Farmacia,
Universitat de Barcelona, 08028 Barcelona, Spain
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ABSTRACT |
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L-1,2-Propanediol:NAD+
1-oxidoreductase of Escherichia coli is encoded by the
fucO gene, a member of the regulon specifying dissimilation
of L-fucose. The enzyme normally functions during fermentative growth to regenerate NAD from NADH by reducing the metabolic intermediate L-lactaldehyde to propanediol which
is excreted. During aerobic growth L-lactaldehyde is
converted to L-lactate and thence to the central metabolite
pyruvate. The wasteful excretion of propanediol is minimized by
oxidative inactivation of the oxidoreductase, an
Fe2+-dependent enzyme which is subject to
metal-catalyzed oxidation (MCO). Mutants acquiring the ability to grow
aerobically on propanediol as sole carbon and energy source can be
readily selected. These mutants express the fucO gene
constitutively, as a result of an IS5 insertion in the
promoter region. In this study we show that continued selection for
aerobic growth on propanediol resulted in mutations in the
oxidoreductase conferring increased resistance to MCO. In two
independent mutants, the resistance of the protein was respectively
conferred by an Ile7 Leu and a Leu8
Val
substitution near the NAD-binding consensus amino acid sequence. A
site-directed mutant protein with both substitutions showed an MCO
resistance greater than either mutant protein with a single amino acid
change.
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INTRODUCTION |
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Oxidative damage to macromolecules is an inescapable price for the
evolution of aerobic metabolism. The co-evolution of protective agents,
both catalytic (enzymes such as superoxide dismutases and catalases)
and stoichiometric (antioxidants such as glutathione and
-tocopherol), can at best reduce the magnitude of damage. Evolution
of active mechanisms of repair apparently is limited only for DNA,
probably because of chemical feasibility and strong selective pressure.
For other kinds of damaged macromolecules clearing by turnover seems to
be the only option. A possible exception is the repair of oxidized
methionine residues on the surface of proteins by a specific sulfoxide
reductase (1). The replacement strategy seems satisfactory for the
perpetuation of unicellular organisms, although it is not always
available or adequate for multicellular organisms. For instance,
accumulation of oxidatively damaged proteins is often associated with
senescence and various disease states (2-4).
A significant fraction of protein damages is thought to result from the metal-catalyzed oxidation system (MCO)1 mediated by reactive species such as hydrogen peroxide, as outlined in Reactions 1 (2, 4, 5).
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When the iron is bound to a protein, the H2O2-dependent redox cycling of Fe2+ to Fe3+ is thought to proceed in a "cage," thus allowing the hydroxyl radical to extract an H atom from a local amino acid residue, before diffusing into the surrounding medium (6-9). Such a model would account for the limited number of amino acid residues that are susceptible to the damage, with each protein exhibiting a distinctive target signature of residues. The model is also supported by the evidence of substrate protection against oxidative damage (6, 8, 10). Although Arg, Cys, His, Lys, Met, and Pro residues are most susceptible to metal-catalyzed destruction, only the oxidation of Arg, Pro, His, and Lys has been reported to result in the formation of a carbonyl derivative which provides a means for monitoring the protein oxidation process (2, 11).
L-1,2-Propanediol:NAD+ 1-oxidoreductase of Escherichia coli is an Fe2+-dependent enzyme that normally functions as a reductase in a fermentation pathway for the dissimilation of L-fucose or L-rhamnose (12). This enzyme, inducible by either of the methyl pentoses, is inactivated during aerobic growth (9, 13, 14). In this study we tried to find out whether repeated selection of mutants that utilize this enzyme exclusively as a dehydrogenase for aerobic growth, on propanediol as the sole carbon and energy source, would result in an altered protein resistant to oxidative inactivation.
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EXPERIMENTAL PROCEDURES |
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Sequencing the fucO Gene of Strains ECL1, ECL3, ECL56, ECL421, ECL430, and ECL459-- The oligonucleotide primers 5'-CGGATCCGGCATTATCACATCAG and 5'-CGAATTCAAGAGTAATTTCGTAAAGC flanking the coding region of fucO (15, 16) were synthesized and used to amplify by polymerase chain reaction the gene of each strain. A sample of each amplified product was digested with restriction enzymes to give five overlapping fragments that were then subcloned into pBluescript vectors (Stratagene, La Jolla, CA). Each of these fragments was sequenced for both strands with the T3 and T7 primers (Stratagene, La Jolla, CA) by the dideoxy method.
Selecting an E. coli Strain with Complete Deletion of the Fuc
Sequence--
Cells with deletions in the fuc locus were
selected from strain ECL330 bearing a fuc::Mu
d1-ampr insertion by growth at a nonpermissive temperature
(42 °C) that allows the growth of only Mu dl cells.
Clones with a Mu dl
Amps Fuc
phenotype were identified by growth at 42 °C on MacConkey/fucose medium and then purified by growing at 37 °C on the same medium. The
deletion ranges in these cells were screened by Southern blots probed
with a set of fuc fragments spanning the entire region of
the fuc regulon (17). A strain completely deleted in the fuc regulon was thus identified and designated as
ECL733.
Generating Mutant Alleles of fucO--
The
fucOIle-7Leu and
fucOLeu-8
Val mutations were regenerated
by site-directed mutagenesis (18) of the wild-type sequence. A
fucO allele with the above two mutations combined,
fucOIle-7
Leu, Leu-8
Val, was created in
parallel. A 5-kb SalIo-BamHI fragment
cut from pfuc16 (19) containing the wild-type fucO allele
was subcloned into an M13 mp18 vector (Stratagene, La Jolla, CA), and
the DNA was used to transfect CJ236 cells (Bio-Rad). Single-stranded
M13 DNA was then prepared from the cells, hybridized to the
oligonucleotide primers containing the desired point mutations (ordered
from Oligos Etc., Inc., Guilford, CT), and used as template for
synthesizing the second DNA strand. The duplex products were used to
transfect XL1 cells (Stratagene, La Jolla, CA). A plaque from each
transfection was purified, and a fucO fragment in the phage
DNA was sequenced to confirm the nucleotide substitution(s). The phage
DNA containing the mutated fucO alleles was respectively designated
FM5/OIle-7
Leu,
FM5/OLeu-8
Val, and
FM5/OIle-7
Leu, Leu-8
Val.
Cloning the Entire Wild-type Fuc Regulon into a Shuffling
Vector--
As shown in Fig. 1, a
modified pBluescript plasmid was used as an intermediary shuffling
vector for carrying fuc sequences onto vector. The
modification was made by inserting a camr gene
cut from a pBC KS+ plasmid (Stratagene, La Jolla, CA) into
the pBluescript polylinker region, to place the cloned fuc
sequence close to a selectable marker for subsequent insertion into the
chromosome via
vectors (see below). The resulting plasmid was
designated pBB. The wild-type fuc regulon (about 9 kb) was
cloned into pBB in a two-step procedure as follows: step 1, an
approximately 8-kb fuc fragment
PvuII-SalI1 was cut from pfuc1
containing the entire fuc regulon (19) and inserted into
pBB; and step 2, the remaining 1-kb fuc fragment Sa1I1-Sa1I2 was appended to the insertion by
substituting the EcoRI-SalI1 fragment
in the first insertion with the
EcoRI-SalI2 fragment cut from pfuc1.
The resulting plasmid was designated pFB1. For the propagation of
various plasmids, XL1 cells were used.
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Cloning Fuc Regulons with the Mutated fucO Alleles into the
Shuffling Vector--
The mutant fuc regulons were cloned
into the shuffling vector by substituting the wild-type fucO
sequence in pFB1 with the mutated counterparts. This procedure was
carried out in eight steps, illustrated in a condensed manner in Fig.
2. Step 1: a pBC KS+ plasmid
was modified by destroying the single PvuI restriction site
with the Klenow enzyme and ligase, resulting in a second shuffling
vector pBC* (not shown). Step 2: a 7-kb fuc
fragment SalI0-EcoRI was cut from
pfuc16 and inserted into pBC*, resulting in pFC1. Step 3: a
2.2-kb fragment BamHI-BamHIlinker was
deleted from pFC1 to eliminate a PstI site in the pBC
polylinker region, resulting in pFC2 (not shown). Step 4: the 0.5-kb
fuc0 fragment PstI-PvuI in pFC2 was
cut off and substituted with a corresponding fragment bearing one of
the fucO mutations cut from FM5/OIle-7
Leu,
FM5/OLeu-8
Val, or
FM5/ OIle-7
Leu, Leu-8
Val, resulting
in pFC2/OIle-7
Leu,
pFC2/OLeu-8
Val, or
pFC2/OIle-7
Leu, Leu-8
Val (not shown).
Step 5: the PstI-PvuI fragments in
pFC2/OIle-7
Leu,
pFC2/OLeu-8
Val, and
pFC2/OIle-7
Leu, Leu-8
Val were
sequenced to confirm the correct substitutions. Step 6: the
BamHI-BamHIlinker fragment from pFC1
was inserted back into pFC2/OIle-7
Leu,
pFC2/OLeu-8
Val, and
pFC2/OIle-7
Leu, Leu-8
Val, resulting in
plasmids pFC1/OIle-7
Leu,
pFC1/OLeu-8
Val, and
pFC1/OIle-7
Leu, Leu-8
Val. Step 7: the
5-kb fuc fragment PstI-EcoRI in pFB1
was cut off and substituted with a corresponding fragment cut from
pFC1/OIle-7
Leu,
pFC1/ OLeu-8
Val, or
pFC1/OIle-7
Leu, Leu-8
Val, resulting in
pFB1/OIle-7
Leu,
pFB1/OLeu-8
Val, or
pFB1/OIle7-
Leu, Leu-8
Val, plasmid bearing
full-length fuc regulon. Step 8: the
PstI-PvuI fragments in
pFB1/OIle-7
Leu,
pFB1/OLeu-8
Val, and
pFB1/OIle-7
Leu, Leu-8
Val were sequenced
to confirm the correct fucO mutation status.
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Inserting Wild-type and Mutant Fuc Regulons into Host
Chromosomes via Vectors--
The plasmids pFB1,
pFB1/OIle-7
Leu,
pFB1/OLeu-8
Val, and
pFB1/OIle-7
Leu, Leu8
Val were digested
with restriction enzymes ApaI and XbaI, yielding
11-kb fragments each containing a full-length fuc regulon
and the camr gene. These fragments were then
ligated with wild-type
DNA cut with the same enzymes. The ligation
mixtures were packaged with the Gigapack Gold Lambda packaging extract
(Stratagene, La Jolla, CA) and used to infect the
fuc
strain ECL733. Cells bearing the foreign sequences on the chromosome
were selected as
lysogens growing on chloramphenicol and
MacConkey/fucose plates. Single copy fuc regulon insertions
were confirmed by Southern blots probed with fuc fragments
at both ends of the fuc sequence. The strains bearing the
wild-type and mutant fuc regulons at the att site are designated ECL734, ECL735, ECL736, and ECL737.
Growth Conditions and Preparation of Cell Extracts-- Cells were grown aerobically as described previously (20) on Luria broth or 0.5% casein acid hydrolysate. Anaerobic cultures were grown as described previously (20) in 1% casein acid hydrolysate supplemented with 1 mM pyruvate. Where indicated, L-fucose was added as inducer at 10 mM concentration for aerobic growth and 20 mM for anaerobic growth. For enzyme assays, cells were harvested at the end of the exponential phase, and cell extracts were prepared as described previously (20) in 10 mM Tris-HCl buffer, pH 7.5. For enzyme purification, the extracts were prepared using a 50 mM Tris-HCl buffer, pH 7.5, containing 2.5 mM NAD.
Enzyme Purification-- Propanediol oxidoreductase was purified from extracts of cells grown anaerobically in Luria broth plus L-fucose by the method of Cabiscol et al. (9). Enzyme purity was assessed by electrophoresis performed according to Laemmli (21) using 10% acrylamide as resolving gel. Proteins were stained with Coomassie Blue R-250.
Enzyme Activity Assays-- Propanediol oxidoreductase was routinely assayed by its NADH-dependent glycolaldehyde reduction to ethylene glycol (22). Glycolaldehyde, readily available commercially, was shown to be an alternative substrate for the enzyme (23). In experiments testing enzyme protection by NAD or propanediol, the enzyme was assayed by NAD-dependent propanediol dehydrogenation (20). Protein concentration was determined by the Bradford method (24) using bovine serum albumin as standard. Immuno-quantification of propanediol oxidoreductase protein was carried out by Laurell rocket immunoelectrophoresis (25) according to a calibration curve (not shown) derived by using the propanediol oxidoreductase purified from strain ECL1. Antibodies were obtained as described (26).
Testing Thermal Stability-- Stock solutions of purified propanediol oxidoreductases (0.5 mg/ml) in 50 mM Tris-HCl buffer at pH 7.5 were kept at 4 °C. At time 0, 0.5 ml of each solution was transferred to tubes preincubated at different temperatures, with or without supplementation with 50 mM DL-1,2-propanediol or 1 mM NAD. Enzyme activities were assayed at various intervals. Thermal stability of the enzyme in crude extract (0.5-ml samples containing 8 mg/ml of total protein) was tested under similar conditions.
Testing Oxidative Inactivation by NADH-- Purified propanediol oxidoreductases (0.5 mg/ml) were incubated at 20 °C in 50 mM Tris-HCl buffer, pH 7.5, in the presence of 0.5 mM NADH. Enzyme activities were assayed at various intervals.
Testing Oxidative Inactivation by Ascorbate Plus Iron-- Purified propanediol oxidoreductases (0.5 mg/ml) were preincubated at 20 °C for 5 min with 50 mM 1,2-propanediol, followed by addition of 15 µM FeCl3 and 30 mM ascorbate according to Levine et al. (27). Enzyme activities were assayed at various intervals.
Immunodetection of Protein Carbonyl Groups-- Dinitrophenylhydrazine derivatization of protein carbonyl groups resulting from oxidation was performed according to Levine et al. (28). Electrophoresis was performed using 10% acrylamide as resolving gel. After electrophoresis the proteins were transferred to polyvinylidene difluoride membranes by semi-dry blotting. Immuno-detection of protein-bound dinitrophenylhydrazones was performed according to Levine et al. (27). The primary antibody was a polyclonal rabbit preparation (Dako, V0401, Denmark). The secondary antibody was goat anti-rabbit conjugated with alkaline phosphatase (Tropix, Bedford, MA).
Metal Analyses--
Atomic absorption spectroscopy measurements
were conducted with a Jobin-Yvon spectrometer, JY-38. Samples were
submitted to high performance liquid chromatography gel filtration in a
Protein Pak 125 (Waters) prior to metal analysis to eliminate reagents and metals not bound to the enzyme. The eluent used was MilliQ water
(resistivity greater that 18 M). Fractions were collected in
metal-free polypropylene tubes.
Fourth Derivative Spectra-- Absorption spectra and their fourth derivative were taken on a Shimadzu UV-160A spectrophotometer, using a derivative interval of 2.4 nm, a slit width of 2 nm, and a scan rate of 80 nm/min. Purified enzymes were dissolved in 50 mM Tris-HCl buffer, pH 7.5.
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RESULTS |
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Wild-type and Propanediol-positive Strains and Comparison of Their fucO DNA Sequences-- Propanediol oxidoreductase is encoded by the fucO gene, a member of the fuc regulon specifying the utilization of L-fucose. The genes of the fuc regulon are organized in two divergent operons, fucPIK and fucAO (Fig. 3). Induction of the operons requires the activator FucR (17, 29) and its effector, L-fuculose-1-phosphate (30). L-1,2-Propanediol, a product of L-fucose fermentation, cannot serve aerobically as a sole carbon and energy source, because the 3-carbon compound fails to induce the fuc regulon expression. If fucO is expressed constitutively at an adequate level, the cell should be able to grow on propanediol by converting it to pyruvate via L-lactaldehyde and L-lactate (Fig. 3).
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Reconstruction of the fucO Mutations in an Otherwise Wild-type Background-- To find out whether the two different single fucO mutations conferred resistance of the enzyme to aerobic inactivation, we tested plasmid-borne wild-type and mutant fucO alleles in a standard wild-type background. Preliminary activity assays of the oxidoreductase were not satisfactorily reproducible. The difficulty seems to be attributable to variations in the plasmid copy number.
To improve the reproducibility of enzyme activity assays, we decided to use strain ECL733 with a complete deletion of the fuc regulon to host aExpression of fucO Genes--
The lysogens ECL734
(fucO+), ECL735
(fucOIle-7
Leu), ECL736
(fucOLeu-8
Val), and ECL737
(fucOIle-7
Leu, Leu-8
Val) and the
wild-type nonlysogen, ECL1, were grown aerobically or anaerobically on
casein acid hydrolysate in the presence of L-fucose as
inducer of the fuc regulon. Extracts from each culture were assayed for propanediol oxidoreductase activity
(Table I). The specific activities of
propanediol oxidoreductase (units/mg enzyme protein) in extracts of
anaerobically grown cells were all about the same, irrespective of the
nature of the fuc allele. In contrast, the specific activity
of the enzyme in extracts of aerobically grown cells was dependent on
the fucO allele: FucO+ < FucOIle-7
Leu < FucOLeu-8
Val < FucOIle-7
Leu, Leu-8
Val. It should be mentioned that
if the inducer was not added, there was no detectable oxidoreductase
activity in extracts prepared from the different strains, even when the
cells were grown anaerobically (not shown), indicating that
transcriptions were from the same fuc promoter. The data
taken together indicate that a single amino acid substitution was
sufficient to confer significant resistance of the protein to oxidative
damage and that the double amino acid substitutions have an additive
protective effect. On the other hand, the amino acid substitutions do
not appear to have a significant effect on the catalytic property of
the enzymes, at least when assayed by the reduction of the substrate
analog glycolaldehyde (Table II).
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Thermal Inactivation of Wild-type and Mutant Propanediol
Oxidoreductase Enzymes--
Additional evidence of structural
alteration of the mutant propanediol oxidoreductase was their decreased
thermal stability. When purified wild-type and mutant propanediol
oxidoreductases were incubated at two different temperatures at pH 7, the activity decay rate of the enzymes followed the same order: at
50 °C, FucO+ (t1/2 = 6.6 min),
FucOIle-7Leu (t1/2 = 2.3 min),
FucOLeu-8
Val (t1/2 = 1.5 min), and
FucOIle-7
Leu, Leu-8
Val (t1/2 = 0.9 min) (Fig. 4A); at
20 °C, FucO+ (t1/2 = 840 min),
FucOIle-7
Leu (t1/2 = 340 min),
FucOLeu-8
Val (t1/2 = 140 min),
and FucOIle-7
Leu, Leu-8
Val (t1/2 = 48 min) (Fig. 4B). It is, however, not clear from these experiments whether the loss of activity resulted from irreversible denaturation of the protein or the loss of the cofactor
Fe2+.
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Oxidative Inactivation of Purified Propanediol Oxidoreductase
Enzymes by NADH--
Since propanediol oxidoreductase is an iron
enzyme, the presence of both molecular oxygen and NADH is expected to
result in an intrinsically catalyzed Fenton reaction that damages the
protein. Purified wild-type and mutant enzymes were incubated for 120 min in the presence of 0.5 mM NADH at 20 °C under air.
As shown in Fig. 5A,
FucO+ was rapidly inactivated (t1/2 = 15 min). In contrast, the mutant proteins were substantially more stable:
FucOIle-7Leu (t1/2 = 28 min),
FucOLeu-8
Val (t1/2 = 54 min), and
FucOIle-7
Leu, Leu-8
Val (t1/2 = 110 min). The possibility that differences in sensitivity of the
enzymes are attributable to the disparities in the amount of iron bound
was excluded by the finding that the metal contents of the four
different purified enzymes were equal, as determined by atomic
absorption spectroscopy. The iron-dependent Fenton reaction as the cause of the observed enzyme inactivation was suggested by the
NADH dependence of oxidative protein damage, as revealed by
immunochemical assays for the carbonyl groups generated (Fig. 5B).
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Oxidative Inactivation of Purified Propanediol Oxidoreductase
Enzymes by Ascorbate and Fe3+--
Other evidence of the
susceptibility of propanediol oxidoreductase to damage by Fenton
reaction was demonstrated by incubation of the protein in the presence
of ferric chloride and ascorbate, instead of NADH, as the reductant
(28). All four enzymes were incubated at 20 °C for 120 min in the
presence of 50 mM DL-1,2-propanediol to
stabilize the proteins against simple thermal inactivation. The
relative rates of inactivation of the four enzymes were consistent with
those observed during incubation with NADH (Fig. 5A):
FucO+ (t1/2 = 70 min),
FucOIle-7Leu (t1/2 = 110 min),
FucOLeu-8
Val (t1/2 = 260 min),
and FucOIle-7
Leu, Leu-8
Val (t1/2 = 430 min) (Fig. 6).
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Analysis of Fourth Derivative Ultraviolet Spectra of Wild-type and
Mutant Enzymes--
On the basis of crystallographic data on the
highly conserved folding motifs of NAD-dependent
oxidoreductases, it can be predicted that the amino acid substitutions
found in the mutant FucO proteins are close to the A-
2 turn of
the mononucleotide-binding motif (or Rossmann fold) where
Tyr31 is located (17, 37).
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DISCUSSION |
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Although early work on propanediol oxidoreductase showed that Fe2+ activates the apoenzyme (41) and that the protein induced during aerobic growth lacked catalytic activity (42, 43), MCO of the enzyme protein was not suspected until a hint was provided by the work on Fe2+-catalyzed inactivation of E. coli glutamine synthetase (6, 7, 44-46). In the case of FucO, the Fe2+ is at the catalytic center which binds the protein to NADH for fermentative reduction of L-lactaldehyde. During aerobic metabolism when the enzyme serves no physiological role, the generation of H2O2 allows the Fe2+ to catalyze a Fenton reaction, and the highly reactive OH· formed is likely to diffuse a distance of the order of its mean free path, which is only a few times of the free radical's radius, before hitting a target amino acid residue (i.e. diffusion-controlled encounter). A frequent occurrence is the destruction of the side chain of a conserved His277, 10 residues away from the proposed metal-binding site, His263-X-X-X-His267, causing a decrease in the apparent affinity of the protein for NAD (13).
It is remarkable that the relatively conservative hydrophobic amino
acid substitutions, Ile7 Leu and/or Leu8
Val, near the NAD-binding sequence
Gly15-Arg-Gly-Ala-Val-Gly20 of FucO (15, 16,
47) could confer significant protective effects against MCO damage.
This resistance, however, is achieved at a price of decrease in protein
stability. The loss of stability might be attributable to "cavity
creation" associated with diminished hydrophobic interactions. In the
case of T4 lysozyme a Leu
Ala substitution raised the
G of the folded form of the enzyme by 1.9 kcal/mol;
increases as high as 6.2 kcal/mol have been observed in hydrophobic
amino acid substitutions in other proteins (48).
Interestingly, the alcohol oxidoreductase II of Zymomonas mobilis, an enzyme highly homologous to FucO (16), is damaged by MCO in a similar way (9). Similar cases of enzyme inactivation by MCO were observed in studies of Klebsiella pneumoniae. Glycerol:NAD 2-oxidoreductase, which serves for the utilization of glycerol under fermentative conditions, is inactivated during aerobic metabolism (49). The initial step involves the MCO-caused loss of apparent affinity for NAD (50, 51). 1,3-Propanediol:NAD 1-oxidoreductase (disposing of NADH by reduction of 3-hydroxypropionaldehyde during fermentative growth on glycerol) and ethanol:NAD oxidoreductase (disposing of NADH by reduction of acetyl-CoA during sugar fermentation) seem to be likewise inactivated (52, 53).
Although protein turnover necessitated by MCO is metabolically costly (44, 54, 55), inactivation of certain enzymes during aerobic respiration can be beneficial in the balance. In the case of FucO, the continued presence of a catalytically active protein during aerobic utilization of L-fucose or L-rhamnose would wastefully deplete both L-lactaldehyde and NADH (56, 57). Viewed from this angle, the bound Fe+2 might be regarded as an adaptive self-destruct mechanism for facilitating the transition from fermentative to aerobic metabolism. The same reasoning should apply to ethanol oxidoreductase. In the case of glycerol oxidoreductase, the rapid inactivation of the enzyme would facilitate the shift from the relatively ineffective anaerobic substrate capturing pathway initiated by the NAD-coupled oxidoreductase to the more avid aerobic substrate scavenging pathway initiated by the ATP-driven kinase (58), a kinetic advantage predictable by the Haldane equation relating Keq to the kcat/Km.
In contrast to NAD(P) enzymes that are involved in fermentative metabolism, those that are needed for both aerobic and anaerobic metabolism, such as glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, and malate dehydrogenase of K. pneumoniae, are resistant to inactivation by oxidative metabolism (53). The same is true for the E. coli NAD-linked L-lactaldehyde dehydrogenase that is required only for aerobic substrate utilization (14). These resistant enzymes are probably dependent on Zn2+ instead of Fe2+ or are metal-independent (see below). An interesting case is the glucose-6-phosphate dehydrogenase in Leuconostoc mesenteroides, which is thought to mediate only anaerobic glucose catabolism by a pentose pathway (59); it is Fe2+-dependent and subject to MCO inactivation (8).
On the basis of amino acid sequence homology, NAD(P)-dependent alcohol dehydrogenases (oxidoreductases) fall into the following three families: (i) long chain Zn2+-dependent, (ii) short chain Zn2+-independent, and (iii) Fe2+-activated (60, 61). Zymomonas mobilis, an O2-tolerant and obligately ethanologenic anaerobe (62), has two alcohol oxidoreductases, one is Fe2+-dependent and the other dependent on Zn2+ (16, 63-65). As one would expect, the Zn2+ enzyme is MCO-resistant, but the Fe2+ enzyme is susceptible to MCO damage (66). It was suggested that having two enzymes with different metal ion requirement would be a nutritional insurance (67). The question of whether the Zn2+ enzyme plays the major role in aerobic ethanologenesis and the Fe2+ enzyme in anaerobic ethanologenesis has not been addressed. The yeast, Saccharomyces cerevisiae, also possesses both Zn2+ and Mn2+ alcohol dehydrogenases (60). The physiological and/or evolutionary basis for employing both metal ions has yet to be explored.
In light of the fact that no Fe2+-dependent oxidoreductase is known to have an aerobic function, it is tempting to suggest that such enzymes evolved early when the global environment was highly reducing and the supply of ferrous iron was abundant. With the emergence of photosynthesis and attendent accumulation of O2, aerobic metabolism developed, and iron became mostly sequestered as insoluble Fe3+ compounds. Fe2+-dependent oxidoreductases were gradually supplanted by Zn2+-dependent ones. Those oxidoreductases that persisted as Fe2+ enzymes did so either because there was a lack of selective pressure to switch to Zn2+ or because retention of Fe2+ actually provided the cell with an adaptive mechanism for thrifty shift from anaerobic to aerobic metabolism.
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ACKNOWLEDGEMENT |
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We thank Yan Zhu for pointing out the proximity of the FucO substitutions to the NAD-binding sequence.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grants GM40993 and GM30693 from the NIGMS and Grant PB94-0829 from the Direcciòn General de Investigacion Cientifica y Técnica, Madrid, Spain. Help from the Comissionat per Universitats i reçerca de la Generalitat de Catalunya" and the "Ajuntament de Lleida" was also received.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.
§ Both authors contributed equally to this article.
** To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1925; Fax: 617-738-7664; E-mail: ELIN{at}WARREN.MED.HARVARD.EDU.
1 The abbreviations used are: MCO, metal catalyzed oxidation; kb, kilobase pair(s).
2 Z. Lu, unpublished observations.
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REFERENCES |
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