Investigating the molecular determinants for substrate channeling in BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls degradation pathway

Article abstract of DOI:10.1021/bi200960j

BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls (PCBs) degradation pathway, cleaves 4-hydroxy-2-oxoacids to pyruvate and an aldehyde. The enzyme complex was shown to exhibit substrate channeling, whereby linear aldehydes of up to 6 carbons long and branched isobutyraldehyde were directly channeled from the aldolase to the dehydrogenase with greater than 80% efficiency. BphI variants G322F, G322L, and G323F were created and were found to block aldehyde channeling. The dehydrogenase cofactor NADH was able to activate the catalytic activity of the aldol cleavage reaction in these variants, suggesting that activation of BphI by BphJ cofactors is not solely due to faster aldehyde release. A G323L variant was able to channel acetaldehyde but not the larger propionaldehyde while the G323A variant was able to channel butyraldehyde but not its isomer isobutyraldehyde, confirming that the restricted channeling of aldehydes in these glycine variants are due to steric blockage of the channel. Substitution of His-20 and Tyr-290 in BphI led to significant reductions in aldehyde channeling efficiencies. A mechanism of substrate channeling involving these two gating residues is proposed.

Full text of DOI:10.1021/bi200960j

Article
pubs.acs.org/biochemistry
Investigating the Molecular Determinants for Substrate Channeling
in BphI−BphJ, an Aldolase−Dehydrogenase Complex from the
Polychlorinated Biphenyls Degradation Pathway
Jason Carere, Perrin Baker, and Stephen Y. K. Seah*
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
S
* Supporting Information
ABSTRACT: BphI−BphJ, an aldolase−dehydrogenase complex from the polychlorinated
biphenyls (PCBs) degradation pathway, cleaves 4-hydroxy-2-oxoacids to pyruvate and an
aldehyde. The enzyme complex was shown to exhibit substrate channeling, whereby linear
aldehydes of up to 6 carbons long and branched isobutyraldehyde were directly channeled
from the aldolase to the dehydrogenase with greater than 80% efficiency. BphI variants
G322F, G322L, and G323F were created and were found to block aldehyde channeling.
The dehydrogenase cofactor NADH was able to activate the catalytic activity of the aldol
cleavage reaction in these variants, suggesting that activation of BphI by BphJ cofactors is
not solely due to faster aldehyde release. A G323L variant was able to channel
acetaldehyde but not the larger propionaldehyde while the G323A variant was able to
channel butyraldehyde but not its isomer isobutyraldehyde, confirming that the restricted
channeling of aldehydes in these glycine variants are due to steric blockage of the channel.
Substitution of His-20 and Tyr-290 in BphI led to significant reductions in aldehyde
channeling efficiencies. A mechanism of substrate channeling involving these two gating
residues is proposed.
he microbial meta-cleavage pathway is important for the
effects to the cell. Furthermore, it was observed that the kcat
value for BphI was increased about 4-fold when nicotinamide
cofactors of the dehydrogenase were present, suggesting that
communication between the enzymes in the complex ensured
efficient coupling of their respective reactions.4
T
biodegradation of aromatic pollutants such as phenols,
xylenes, and polychlorinated biphenyls (PCBs) and for host
cholesterol utilization by the human pathogen, Mycobacterium
tuberculosis.1−3 These diverse aromatic compounds can be
transformed in the pathway to form a 4-hydroxy-2-oxoacid
intermediate. Carbon bond cleavage between the C3 and C4 of
this intermediate by an aldolase leads to the formation of
pyruvate and an aldehyde.4 The latter is then converted to an
acyl-CoA by an acylating acetaldehyde dehydrogenase. In the
polychlorinated biphenyls degradation pathway of Burkholderia
xenovorans LB400, the class II aldolase BphI forms a stable
complex with the dehydrogenase, BphJ. Previously, using a
substrate competition assay, it was demonstrated that
acetaldehyde and propionaldehyde produced from the BphI-
catalyzed cleavage of 4-hydroxy-2-oxopentanoate (HOPA) and
4-hydroxy-2-oxohexanoate (HOHA), respectively, are not
released to the bulk solvent but are channeled directly to
BphJ.4 Thus, when excess propionaldehyde was added
exogenously to a reaction mixture containing 4-hydroxy-2-
oxopentanoate, 95% of the CoA esters produced were acetyl-
CoA. Conversely, 99% of the CoA esters produced were
propionyl-CoA when an excess of exogenous acetaldehyde was
added in an enzyme reaction mixture containing 4-hydroxy-2-
oxohexanoate. The Km values for exogenous aldehydes in BphJ
are >8 mM, and coupled to the fact that aldehydes are volatile,
reactive, and toxic, channeling and sequestering of aldehydes
from the cytoplasm of the bacterium may be necessary for
efficient conversion to CoA esters and to prevent detrimental
The crystal structure of orthologous aldolase−dehydrogenase
complex (DmpG-DmpF; 56% and 55% sequence similarity to
BphI and BphJ, respectively) from the phenol degradation
pathway of Pseudomonas putida CF600 depicts a ∼21 Å long
hydrophobic tunnel between the aldolase and the dehydrogen-
ase active sites, providing a possible route for the direct
diffusion of aldehyde from the aldolase to the dehydrogenase.5
The presence of a conduit for the passage of common
intermediates in sequential enzyme reactions has been observed
in several other enzyme systems that exhibit substrate
channeling, including tryptophan synthase, carbamoyl phos-
phate synthetase, and glucosamine 6-phosphate synthase.6−8
Unique to the aldolase−dehydrogenase system of the aromatic
degradation pathway is that the respective enzymes have broad
substrate specificities and are capable of utilizing larger
substrates.9,10 While it has been demonstrated that acetalde-
hyde and propionaldehyde can be channeled by the enzyme
complex, it was unclear if longer and branched aldehydes could
also be channeled. This has some practical relevance since the
meta-cleavage pathway for degradation of certain phenols with
Received: June 23, 2011
Revised: August 11, 2011
Published: August 12, 2011
© 2011 American Chemical Society
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Figure 1. Structure of DmpG−DmpF showing (A) the tetrameric arrangement of the biological unit with aldolases colored blue and dehydrogenases
colored green and (B) the location of the tunnel from the aldolase active site to the bulk solvent (blue) and the channel connecting the active sites of
the aldolase to the dehydrogenase (magenta). The location of the tunnel and channel were determined using MOLE.17 Images were generated using
PyMOL.18
alkyl substitutions at the C4 position has been reported in
bacteria, yielding aldehydes up to 7 carbon atoms in length.11,12
By a combination of molecular modeling and site-specific
mutagenesis, we explored the contribution of residues in the
enzyme complex that govern channeling specificity and
efficiency. We developed a method to quantify the efficiency
of aldehyde channeling in the BphI−BphJ complex which
enabled us to determine channeling efficiencies of aldehydes of
varying sizes and lengths. Finally, the creation of channel
blocked enzyme variants enabled us to test if the observed 4-
fold activation of BphI by BphJ when nicotinamide cofactor is
bound is due to faster aldehyde release by substrate channeling.
was generated using ClustalX15 and visualized using ESPript.16
Channels in the orthologous DmpG-DmpF complex from P.
putida CF600 (PDB 1NVM)5 were identified using MOLE17
and visualized in PyMOL.18
Expression and Purification of the Enzyme Complex.
Wild-type and variant BphI−BphJ enzyme complexes were
expressed and purified as previously described.4 Sodium
dodecyl sulfate−polyacrylamide gel electrophoresis was per-
formed and gels stained with Coomassie Blue to assess purity.
Protein concentrations were assessed by Bradford Assay using
bovine serum albumin as the standard.19
Steady-State Kinetic Assays. Aldolase cleavage activity
was determined by coupling the production of pyruvate to
NADH oxidation by LDH.4 All assays were performed at least
in duplicate, at 25 °C in a total volume of 1 mL using a Varian
Cary 3 spectrophotometer with a thermojacketted cuvette
holder. A standard assay contained 0.4 mM NADH, 1 mM
MnCl2, and 19.2 units LDH in 100 mM HEPES buffer (pH
8.0). The concentration of 4-hydroxy-2-oxoacids was varied
from 0.1Km to 5Km. Oxidation of NADH was monitored
continuously at 340 nm with the extinction coefficient taken as
6200 M−1 cm−1. To assess the activity of BphI and variants in
the absence of BphJ cofactors, a discontinuous assay was
utilized. Assay mixtures contain 1 mM MnCl2, HOPA
concentrations varied from 0.1Km to 5Km, and 18 μg of
BphI−BphJ in 100 mM HEPES buffer (pH 8.0). After 10 min,
the reaction was quenched with 20 mM EDTA (pH 8.5) and
0.4 mM NADH. The amount of pyruvate produced was
measured by an end-point assay with LDH.
The activity of the dehydrogenase, BphJ, was monitored
continuously at 340 nm to observe the formation of NADH.
Assays contained 0.4 mM NAD+ and 0.1 mM coenzyme A in
100 mM HEPES buffer (pH 8.0) with varied aldehyde
concentrations from 0.1Km to 5Km. Kinetic parameters were
determined as previously described, and results were fitted by
nonlinear regression to the Michealis−Menten equation using
Leonora.4,20
MATERIALS AND METHODS
■
Chemicals. Sodium pyruvate, LDH (rabbit muscle), and all
aldehydes were from Sigma-Aldrich (Oakville, ON). Restriction
enzymes, T4 DNA ligase, and Pfu Turbo polymerase were from
Stratagene (Mississauga, ON) or New England Biolabs
(Pickering, ON). Ni-NTA Superflow resin was obtained from
Qiagen (Mississauga, ON). 4-Hydroxy-2-oxoacids were synthe-
sized using BphI or HpaI by previously described methods.9
Aldehyde dehydrogenase from Saccharomyces cerevisiae was
from Calzyme (San Luis Obispo, CA). All other chemicals were
analytical grade and were obtained from either Sigma-Aldrich
and Fisher Scientific (Nepean, ON).
Site-Directed Mutagenesis. DNA was purified using
standard protocols.13 bphI and bphJ were previously cloned
into the plasmids pBTL4-T7 and pET28a, respectively.4 Site
directed mutagenesis was performed using a Quickchange
(Stratagene) method for I195L and I195F and a modified
method14 with nonoverlapping primers (Table S1) for other
variants. Genes were sequenced at the Guelph Molecular
Supercenter to confirm that the desired mutations were
incorporated and the absence of secondary mutations. The
L89A, H20A, and Y290F variants were created as previously
reported.10
Structure Modeling and Sequence Alignment. A
multiple-sequence alignment containing orthologous aldolases
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Channeling Assays. The amount of aldehyde channeled
directly from the aldolase to the dehydrogenase was assessed
using an enzyme competition assay. All assays contained 0.4
mM NAD+, 0.1 mM coenzyme A, 1 mM MnCl2, 5 μg of
enzyme (200 μg for the H20A and Y290F), and 20 U of
aldehyde dehydrogenase (ALDH) in a total volume of 1 mL in
100 mM HEPES buffer pH 8.0. Reactions were initiated by
addition of substrate, and the amount of NADH produced was
measured spectrophotometrically at 340 nm. HOPA and
HOHA concentrations were 25 μM for wild-type enzyme and
G322, G323, and L89A variants while 200 μM was used for the
less active variants, H20A and Y290F. Other 4-hydroxy-2-
oxoacids concentrations were used at concentrations of 100
μM.The reaction was quenched after 5 min with 3 N HCl to a
final concentration of 70 mM. The sample was centrifuged for 3
min at 21000g to pellet the denatured enzyme. A 500 μL
sample of the reaction mixture was subjected to high
performance liquid chromatography (HPLC) using an AKTA
Explorer 100 (Amersham Pharmacia Biotech, Baie d’Urfe, QC)
equipped with a HyPURITY C18 column (Thermo Scientific).
Samples were eluted with 50 mM sodium phosphate (pH
5.3)−acetonitrile mixtures at the following ratios: 47:3 for
acetyl-CoA and propionyl-CoA, 125:16 for butyryl-CoA and
iso-butyrl-CoA, 25:4 for pentanoyl-CoA and hexanoyl-CoA.
The flow rate was 1.0 mL/min, and CoA-esters were detected
by UV absorbance at 254 nm. Quantification of the acyl-CoA
molecules produced was completed using standard curves of
the pure compounds. Pentanoyl-CoA standards are not
commercially available and were synthesized using BphJ,
pentaldehyde, CoA, and NAD+. The channeling efficiency
was calculated by comparing the acyl-CoA produced
(determined using HPLC) to the concentration of 4-hydroxy-
2-oxoacid utilized by BphI based on the amount of NADH
produced by BphJ and ALDH (eq 1).
Scheme 1. Cleavage of 4-Hydroxy-2-oxoacid to Pyruvate and
an Aldehyde
a
a
The aldehyde is then channeled to BphJ where an acyl-CoA is
produced using NAD+ and CoA as cofactors. R = CH3 in the biphenyl
degradation pathway.
Figure 2. Model of 4-hydroxy-2-oxononanoate bound in the DmpG
active site. The model was generated by superimposing the pyruvyl
moiety of substrate shown in sticks within the active site on the
experimentally observed oxalate. The tunnel leading from bulk solvent
is shown in blue, and the active site and channel connecting the
aldolase active site to the dehydrogenase is shown in magenta. His-21
blocks the tunnel leading from the bulk solvent. Residues investigated
in this study are shown in sticks. Carbon atoms of Tyr-291 in the
crystal structure is shown in yellow lines and the residue rotated 14°
clockwise about the Cα to avoid steric slash with the C4 of the
modeled substrate is shown in gray as sticks. Leu-90, Gly-323, and Gly-
324 line the narrowest point of the tunnel. Corresponding residue
numbers in BphI are indicated in brackets. The distal end of 4-
hydroxy-2-nonanoate is too long to fit in the active site and is propose
here to protrude into the channel.
(1)
RESULTS
■
Structural Analysis of the Channel Linking Active
Sites of the Aldolase and Dehydrogenase. The crystal
structure of the BphI−BphJ complex is not available; therefore,
structural analysis was performed on the crystal structure of the
orthologous enzymes, DmpG−DmpF, from P. putida CF600
(PDB 1NVM). The asymmetric unit in the crystal consists of
two tetramers, with all aldolases (DmpG) containing bound
Mn2+ and oxalate, a pyruvate enolate analogue, and three of the
four dehydrogenase subunits (DmpF) containing bound NAD+
(Figure 1A). The biological unit of the complex is a tetramer
consisting of two DmpG subunits in the middle and each of the
two DmpF subunits associating with each DmpG subunit at the
periphery to form an elongated structure.5 It has previously
been determined by gel filtration that BphI−BphJ also forms a
tetramer.4 The position of the tunnel linking the bulk solvent to
the active site and the channel linking the aldolase and
dehydrogenase were mapped using the software MOLE using
the crystal structure of DmpG−DmpF as template (Figure 1B).
A narrow tunnel connects the active site of the aldolase,
DmpG, to the bulk solvent, which is presumably the entrance
for 4-hydroxy-2-oxoacid substrates and the exit for the pyruvate
product (Figure 2). The narrowest point of the tunnel
connecting the active sites of DmpG−DmpF (1.6 Å in
diameter) is insufficient in size to allow for passage of 4-
hydroxy-2-oxoacids, and therefore rotation of the proposed
catalytic base, His-21, is assumed to be required to allow for
access of substrates.5 Upon substrate entry, His-21 is proposed
to revert back to its conformation seen in the “closed” position
in order to be of optimal distance and geometry relative to the
C4-OH of the substrate to be effective as a catalytic base.10
Substrate binding is presumably also coordinated by a rotation
about the Cα of Tyr-291 to relieve a steric clash between the
hydroxyl oxygen of Tyr-291 and C4 of the modeled substrate.10
Movement of Tyr-291 would result in an enlargement in the
opening of the channel. Interestingly, when the aldehyde
moiety of the 4-hydroxy-2-oxoacids is longer than 4 carbons in
length, there is no space available in the active site to
accommodate the longer alkyl chain unless the distal end of the
molecule protrudes into the aldehyde channel (Figure 2).
The channel connecting the active sites of DmpG and DmpF
is contributed almost entirely by the aldolase and residues that
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Figure 3. Sequence alignment of BphI and orthologes. Amino acid sequences of proteins and their uniprot accession number are as follows: DmpG
from Pseudomonas putida CF600 (P51016), BphI from B. xenovorans (P51015), BphF from Pseudomonas sp. strain KKS102 (P51014), TodH from
Pseudomonas putida F1 (P51018), CmtG from Pseudomonas putida F1 (Q51983), PhnJ from Pseudomonas sp. Strain DJ77 (Q9Z3U6), CumF from
Pseudomonas fluorescens IP01 (P97092), NahM from Pseudomonas putida (P51017), XylK from Pseudomonas putida (P51019), MhpE from
Escherichia coli (P51020), and LapG from Pseudomonas alkylphenolia (Q7WYE3). Secondary structures of DmpG are indicated as horizontal arrows
(beta strands) and helices (α helix) above the sequences. Conserved residues that line the aldehyde channel are highlighted in red. Residues in BphI
that were replaced by site-directed mutagenesis in this study are indicated by stars at the bottom of the alignment. This alignment was generated
using ClustalX15 and visualized using ESPript.16
compose the channel are conserved in orthologs, including
BphI (Figure 3). The diameter of the aldehyde channel ranges
between approximately 2.4 and 5.4 Å (Figure 4), with the
narrowest point (diameter ∼2.4 Å) lined by residues Gly-323
and Gly-324 on one side and the Cδ1 of Leu-90 on the
opposite wall of the tunnel formed by the aldolase, DmpG.
Together with the side chain of Tyr-291, the Cδ1 of Leu-90
creates the entrance of the aldehyde channel in the aldolase
while the Cδ2 of Leu-90 forms one boundary of the active site
of the aldolase (Figure 2). The exit of the channel, on the other
hand, is formed by three residues of the dehydrogenase DmpF:
Ile-172, Ile-196, and Met-198. These three residues adopt
alternative conformations within and between the ligand free
and ligand bound structures. In the absence of NAD+, these
residues block access to the active site of the dehydrogenase
from the channel. However, in the NAD+-bound form of the
dehydrogenase, movement of the Ile-172 and Met-198 side
chains results in a small opening of the exit of the channel
(diameter ∼1.7 Å).
To probe the contributions of specific residues in aldehyde
channeling, several variants of the BphI−BphJ complex were
created. Gly-322 and Gly-323 (Gly-323 and Gly-324 of DmpG)
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Table 1. Steady-State Kinetic Constants of BphI and
a
Variants for the Aldol Cleavage of HOPA and HOHA
substrate
HOPA
kcat (s−1
HOHA
enzyme
Km (μM)
)
Km (μM)
kcat (s−1
3.9 ± 0.2
)
WT
89 ± 8
140 ± 10
160 ± 20
90 ± 10
940 ± 80
180 ± 20
60 ± 10
4.07 ± 0.07
1.09 ± 0.05
0.64 ± 0.03
1.06 ± 0.04
4.3 ± 0.2
117 ± 10
350 ± 20
370 ± 70
300 ± 20
1100 ± 200
520 ± 90
360 ± 30
G322L
G323L
G322F
G323F
G322A
G323A
0.94 ± 0.02
0.27 ± 0.02
1.05 ± 0.03
1.3 ± 0.1
2.4 ± 0.1
0.34 ± 0.02
1.9 ± 0.1
0.46 ± 0.07
a
Aldolase assays were performed at 25 °C and contained 0.4 mM
NADH, 1 mM MnCl2, and 19.2 units LDH in 100 mM HEPES buffer
(pH 8.0).
Table 2. Steady-State Kinetic Parameters of BphJ and
Variants toward Acetaldehyde and Propionaldehyde
Figure 4. Graphical representation of the diameter of the aldehyde
channel as determined using the program MOLE.17 The tunnel was
mapped from the position of the divalent metal cofactor in the aldolase
to the putative catalytic cysteine in the dehydrogenase. Positions in the
graph corresponding to the aldolase and dehydrogenase active sites are
depicted in blue and green, respectively. Computation of the structure
when Tyr-291 is in the “closed” conformation as in the crystal
structure is shown as a dotted line. The “open” conformation as
predicted by the model is a solid line. The first channel diameter
minima at about 7 Å distance corresponds to the location of the two
glycine residues (323 and 324 in DmpG) that line the channel.
a
substrate
acetaldehyde
Km (mM)
kcat (s−1
propionaldehyde
Km (mM) )
kcat (s−1
enzyme
)
WT
24 ± 2
35 ± 3
7.4 ± 0.5
17.2 ± 0.5
16 ± 1
42 ± 2
23 ± 2
48 ± 9
7.7 ± 0.7
16.3 ± 0.5
8.9 ± 0.8
46 ± 2
I195F
I195L
a
Dehydrogenase assays were performed at 25 °C and contained 0.4
mM NAD+ and 0.1 mM coenzyme A in 100 mM HEPES buffer (pH
8.0).
were replaced with bulkier residues alanine, leucine, and
phenylalanine. Leu-89 of BphI (Leu-90 in DmpG) was replaced
with a smaller alanine residue. This set of variants was created
to probe the effects of reducing or increasing the channel
diameter on aldehyde channeling efficiencies. The contribution
the proposed gating residues in aldehyde channeling was tested
by replacement of His-20 of BphI (His-21 of DmpG) with
alanine and Tyr-290 (Tyr-291 in DmpG) with phenylalanine.
Ile-195 of BphJ (Ile-196 in DmpF) that forms the exit of the
channel was replaced with an isosteric leucine residue and the
larger phenylalanine residue.
Site-Specific Mutagenesis, Protein Purification, and
Steady-State Kinetic Analysis. All variants were successfully
created by site specific mutagenesis and purified using the same
conditions as wild-type4 enzyme with similar yields and purities
achieved. Aldolase kinetic parameters of Y290F and H20A
variants were previously reported,10 and their catalytic
efficiencies were lowered by ∼3-fold and ∼100-fold,
respectively, relative to the wild-type aldolase. Steady-state
kinetics of both the aldolase and dehydrogenase of other
variants were determined and are of the same order of
magnitude as the wild-type enzymes (Tables 1 and 2).
Determination of Aldehyde Channeling Efficiency in
the Wild-Type BphI−BphJ Complex. Previously, using a
substrate competition assay, it was demonstrated that the
enzyme complex can channel acetaldehyde and propionalde-
hyde.4 To obtain a quantitative measure of channeling
efficiency, an enzyme competition assay was developed. Excess
exogenous aldehyde dehydrogenase was added to the assay so
aldehyde released to the bulk solvent would be oxidized to a
carboxylic acid. In contrast, aldehydes channeled to BphJ will
be converted to acyl CoA esters. The amount of acyl-CoA
produced by the enzyme complex in relation to the total
amount of aldehyde produced from the cleavage of 4-hydroxy-
2-oxoacid by BphI provides a quantitative measure of efficiency
of channeling for each aldehyde as indicated by eq 1.
Channeling efficiency is defined here as the amount of
aldehyde channeled from BphI which is utilized by BphJ to
form CoA esters. It also depends on the degree of coupling
between the reactions catalyzed by the aldolase and the
dehydrogenase and represents a lower threshold for the true
amount of aldehyde which traverses the channel.
Using this assay, channeling efficiency for acetaldehyde and
propionaldehyde were determined to be 95 ± 5% and 95 ± 4%,
respectively. Longer straight chain aldehydes up to six carbons
in lengths were also tested and found to be efficiently
channeled by the enzyme complex (Table 3). Surprisingly,
the branched chain aldehyde, isobutyraldehyde, was also
channeled with an efficiency similar to that of acetaldehyde,
the physiological substrate of BphJ.
Role of Internal Channel Residues Gly-322, Gly-323,
and Leu-89 in Aldehyde Channeling Efficiency and
Specificity. Variants G322A and G323A were able to channel
acetaldehyde with similar efficiencies to wild-type (Table 4).
However, channeling efficiencies progressively decreased with
increasing aldehyde chain length. The G322A variant was able
to channel isobutyraldehyde, albeit at a 30% lower efficiency
than the wild-type enzyme, but G323A variant was unable to
channel this branched chain aldehyde. Channeling efficiency for
acetaldehyde was lower in the G323L variant (63% efficiency),
and this variant was unable to channel the larger propionalde-
hyde. The G322L, G322F, and G323F were all unable to
channel either acetaldehyde or propionaldehyde. The kinetic
parameters of the associated dehydrogenases in these variants
were similar to the wild-type enzyme; thus, the reduced
channeling efficiencies were not due to the compromised
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a
Table 3. Channeling Efficiencies of Various Aldehydes in Wild-Type BphI−BphJ Enzyme Complex
channeled aldehyde
acetaldehyde
(CH3CHO)
propionaldehyde
(CH3CH2CHO)
butyraldehyde
isobutyraldehyde
pentaldehyde
hexaldehyde
structure
channeling
efficiency (%)
(CH3(CH2)2CHO)
((CH3)2CHCHO)
(CH3(CH2)3CHO)
(CH3(CH2)4CHO)
95 ± 5
95 ± 4
83 ± 2
92 ± 3
94 ± 4
84 ± 8
a
Assays were performed at 25 °C and contained 0.4 mM NAD+, 0.1 mM coenzyme A, 1 mM MnCl2, and 5 μg BphI−BphJ in the presence of excess
ALDH. Substrate concentration was 25 μM HOPA or HOHA and 100 μM for all other 4-hydroxy-2-oxoacids. CoA esters were detected at 254 nm
by HPLC.
Table 4. Aldehyde Channeling Efficiencies of BphI Variants−BphJ Complexes
channeling efficiency (%)
BphI variant
acetaldehyde
propionaldehyde
butyraldehyde
isobutyraldehyde
pentaldehyde
hexaldehyde
G322A
G323A
G322L
G323L
G322F
G323F
L89A
89 ± 1
95 ± 10
0
62 ± 7
58 ± 1
0
74 ± 2
29 ± 3
60 ± 3
0
62 ± 1
0
0
0
0
0
67 ± 3
68 ± 1
79 ± 2
55 ± 2
67 ± 6
17 ± 1
activity of BphJ (Table S2). Creating a channel with a larger
diameter by replacing Leu-89 located on the opposite side of
Gly-322 and Gly-323 to alanine led to a surprising reduction in
channeling efficiency of 30% for all aldehydes tested. The
reduction of channeling efficiency in the variant could be due to
the increased size of the channel entrance, subverting the gating
mechanism, leading to escape of the aldehyde from the
substrate entry/pyruvate exit tunnel.
(Table 6). The kcat for the aldol cleavage of HOPA in the
G322L, G322F, and G323F variants, which are unable to
Table 6. Steady-State Kinetic Constants of BphI and Glycine
Variants for the Aldol Cleavage of HOPA in the Absence of
a
BphJ Cofactors
fold activation of BphI by
b
enzyme
Km (μM)
kcat (s−1
)
NADH
Role of Ile-195, Tyr-290, and His-20 in Aldehyde
Channeling. In contrast to the glycine variants, substitution
of Ile-195 in BphJ to a bulkier phenylalanine residue did not
significantly reduce the channeling efficiency of the enzyme
complex toward acetaldehyde or propionaldehyde (Table 5).
WT
158 ± 20
81 ± 14
216 ± 7
53 ± 13
130 ± 10
0.79 ± 0.06
0.286 ± 0.02
0.100 ± 0.009
0.45 ± 0.07
0.72 ± 0.02
5.1 ± 0.4
3.8 ± 0.3
6.4 ± 0.6
2.4 ± 0.2
6.0 ± 0.2
G322L
G323L
G322F
G323F
a
Assays contained 1 mM MnCl2, HOPA concentrations varied from
Table 5. Channeling Efficiencies of Y290F and H20A
Variants of BphI and I195 Variants in BphJ
b
0.1Km to 5Km in 100 mM HEPES buffer (pH 8.0). Ratio of kcat of
BphI in the presence of NADH to the kcat in the absence of NADH.
BphI variants are in complex with wild-type BphJ.
channeling efficiency (%)
enzyme
acetaldehyde
propionaldehyde
channel aldehyde, were still higher by about 3−6-fold in the
presence of NADH. This indicates that faster aldehyde release
in the aldolase due to aldehyde channeling is not solely
responsible for the activation of BphI by BphJ.
I195L (BphJ)
I195F (BphJ)
Y290F (BphI)
H20A (BphI)
79 ± 7
80 ± 1
57 ± 2
11 ± 3
94 ± 1
83 ± 5
38 ± 1
15 ± 1
DISCUSSION
■
On the other hand, substitutions of the Tyr-290 in BphI with
the smaller phenylalanine residue reduced channeling efficien-
cies by >30%. Replacement of His-20, a residue that gates the
substrate entry tunnel, with alanine dramatically reduced
acetaldehyde and propionaldehyde channeling by more than
70%.
Activation of Aldolase by Dehydrogenase in the
Channeled Blocked Variant. On the basis of the crystal
structure of the DmpG−DmpF complex, binding of
nicotinamide induces the opening of the exit of the channel
in the dehydrogenase, thus facilitating aldehyde channeling. We
previously observed that BphI activity is activated 4-fold in the
presence of NADH. To test if this activation is due to allosteric
regulation of BphI by BphJ or simply faster product (aldehyde)
release due to channeling, kinetic parameters for the glycine
variants that block aldehyde channeling were determined
Substrate channeling involves the transfer of a common
intermediate from one enzyme to another without release
into bulk solvent.21 In the case of the aldolase−dehydrogenase
complex in aromatic degradation pathways, sequestering of
volatile aldehydes from cellular components is beneficial
because it prevents toxicity associated with reactive aldehydes
and overcomes the inefficiency of the dehydrogenase for
processing exogenous aldehydes due to high Km values.
Probing the Route and Diameter of the Channel.
Analysis of the DmpG−DmpF crystal structure showed that
residues forming the channel that link the active sites of the
aldolase and dehydrogenase are predominantly provided by the
aldolase and are highly conserved in orthologous complexes.
The molecular dimensions of the channel in the aldolase−
dehydrogenase complex, with a narrowest diameter of 2.4 Å,
appear to be incompatible for passage for the channeled
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aldehyde (4.3 Å). This suggests that the channel is not rigid but
dynamic movement of residues lining the channel is required to
allow the intermediate to pass through. While the low B-factors
of the channel in the crystal structure of DmpG−DmpF can
indicate rigidity, this may be due to crystal packing constraints.
Alternatively, since the structure does not contain aldehyde in
the channel, it does not represent the complex during substrate
channeling. Protein dynamics have also been suggested to occur
in other enzyme systems that exhibit substrate channeling,
including glutamine-dependent NAD+ synthetase22 and proline
utilization A flavoenzyme (PutA).23 In each case constrictions
were observed in the structure of the channel, and therefore
significant movement of residues in the channels is required to
enable the passage of the channeled intermediate.
It has been determined previously that BphI and BphJ have
broad substrate specificity,4,10 although preceding enzymes in
the PCBs degradation pathway have not been shown to utilize
substrates that will lead to formation of compounds larger than
4-hydroxy-2-oxohexanoate. It is therefore surprising that
aldehydes up to 6 carbons in length are channeled from the
aldolase to the dehydrogenase with comparable channeling
efficiency to that of acetaldehyde. The fact that the branched
chain isobutyraldehyde can also be channeled by the complex
indicates that these straight-chain aldehydes need not
necessarily adopt an extended conformation in order to
transverse from the aldolase to the dehydrogenase. Flexibility
of the tunnel of carbamoyl phosphate synthetase has also been
suggested to account for the ability for the enzyme to channel
the larger hydrazine and hydroxylamine in addition to its
natural channeled substrate, ammonia,24 although the efficiency
of channeling of these larger amines relative to ammonia has
not been quantified.
Evidence that the putative tunnel identified from the crystal
structure is indeed the route taken by channeled aldehydes was
supported by the results from replacements of two residues,
Gly-322 and Gly-323, which line the narrowest diameter of the
channel. The G322L and G322F variants completely abrogate
substrate channeling, suggesting that these residues have
effectively blocked the channel while the G322A variant
displayed ∼20% reduced aldehyde channeling efficiencies
relative to the wild-type complex. On the other hand, as the
side-chain size at position 323 was increased, the size of the
aldehyde that these variants could efficiently channel became
progressively smaller, supporting the premise that lower
channeling efficiencies observed in the variants are due to
steric blockage of the channel. In tryptophan synthase, physical
blockage of indole channeling has also been attempted by
replacing a cysteine residue (Cys-170) with tryptophan and
phenylalanine, resulting in reduced indole channeling rate by at
least 10- and 5000-fold, respectively.25 In carbamoyl phosphate
synthetase, replacement of Gly-359 with phenylalanine or
tyrosine residues blocked ammonia channeling, manifested by
uncoupling of hydrolysis of glutamine to carbamoyl phosphate
formation and a lag in the time course for carbamoyl phosphate
formation.24 A G359S variant in carbamoyl phosphate
synthetase exhibited reduced ammonia channeling while
preventing hydroxylamine channeling while the G359L variant
is defective in channeling both compounds.
the channeling of the aldehyde prior to the release of pyruvate.
A reduction in aldehyde channeling efficiency was observed
when two residues His-20 and Tyr-290, present at the substrate
entrance and aldehyde channel entrance, were replaced by
alanine and phenylalanine, respectively. It appears that these
residues may be imperative in ensuring the coordinated release
of products and preventing escape of acetaldehyde from the
substrate entrance/pyruvate release site in BphI. When His-20,
the proposed catalytic base of the enzyme, is poised for C4-OH
proton abstraction of the substrate, the substrate entry/
pyruvate release tunnel that leads from the aldolase active site
to the bulk solvent is closed. This presumably prevents
aldehyde from escaping through this route following C−C
bond cleavage of the substrate. The H20A variant is still active,
albeit with a 45-fold lowering of kcat, due to hydroxide ions
replacing the function of the catalytic base in the variant.10 The
variant however displayed a marked reduction (>70%
reduction) in aldehyde channeling efficiency, possibly due to
the fact that the methyl side chain of alanine is insufficient in
size to effectively block the escape of aldehydes from the
aldolase. In the proline dehydrogenase, PutA, which channels a
pyrroline-5-carboxylate intermediate, a tyrosyl residue (Tyr-
437) has been similarly observed to block the active site of the
dehydrogenase domain from the bulk solvent.26 This residue
could potentially function as a substrate entry gate analogous to
that of His-20 in BphI.
We previously demonstrated that the invariant tyrosine
residue in the active site of BphI is not the catalytic acid,
contrary to a previously proposed mechanism which suggested
that during proton transfer torsional movement of Tyr-290
results in an opening of the channel entrance allowing for
aldehyde channeling to the dehydrogenase to occur.10 Closer
inspection of the active site of the aldolase suggests that the
binding of the modeled 4-hydroxy-2-oxoacid substrate will
result in a steric clash between the C4 of the substrate and the
oxygen in the para position of the phenol ring, necessitating the
movement of this residue upon substrate binding which could
lead to the opening of the tunnel for aldehyde channeling prior
to the aldol cleavage reaction.10 Upon cleavage of the 4-
hydroxy-2-oxoacid, the aldehyde is released into the tunnel and
Tyr-290 may adopt a closed conformation, as visualized in the
crystal structure of DmpG−DmpF, preventing the back-
diffusion of the aldehyde. In the BphI Y290F variant,
channeling efficiency was reduced by about 30%, and this
could be due either to relief of the steric constraint that is
required for tunnel entrance opening or to the fact that the
tunnel cannot be closed completely, allowing some aldehydes
to escape from the aldolase through the substrate entry/
pyruvate release site. Aromatic residues have been proposed as
gating residues in other enzyme systems that exhibits substrate
channeling. For example, the indole side chain of Trp-74 was
observed to adopt an alternative rotameric conformation that
opens the ammonia tunnel when a glutamine analogue was
bound in the enzyme.27 In tryptophan synthase movement of
Phe-280 in the presence of K+ and Cs+ results in opening of the
indole channel.28
When Leu-89 in BphI was replaced with the smaller alanine
residue, aldehyde channeling efficiencies were also reduced.
The delta carbons of Leu-89 form two faces of the tunnel. Cδ1
is directly opposite to Gly-322 and Gly-323 while Cδ2 forms
the base of the active side of the aldolase. Replacing Leu-89
with alanine increases the size of the active site of the aldolase
and should broaden the aldehyde channel. Enlargement of the
Channel Entrance and Exit Residues. Previous steady-
state kinetic analysis of BphI indicated that the enzyme follows
an ordered sequential mechanism whereby the acetaldehyde
product must leave the active site first prior to the release of the
other product, pyruvate.9 Intuitively this is important to allow
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Figure 5. Proposed mechanism of substrate channeling in BphI−BphJ. (A) 4-Hydroxy-2-oxopentanoate (HOPA) enters the aldolase through the
substrate tunnel where it passes His-20. (B) Upon substrate entry, Tyr-290 rotates to accommodate the substrate in the active site, thus opening the
aldehyde channel. His-20 adopts a closed conformation and abstracts a proton from the C4-OH of the substrate to initiate the aldol cleavage
reaction. (C) Upon aldol cleavage, the acetaldehyde is channeled to the dehydrogenase via the aldehyde channel and Tyr-290 reverts to a “closed”
position, preventing the aldehyde from diffusing back into the aldolase active site. (D) His-20 adopts an “open” conformation, allowing pyruvate to
leave and a second molecule of substrate to enter.
aldolase active site in the L89A variant has been demonstrated
to increase the specificities of the aldolase for butyraldehyde,
pentaldehyde, and hexaldehyde while maintaining similar
specificities for acetaldehyde and propionaldehyde.10 However,
this modification had negative effects for aldehyde channeling,
suggesting that the channel cannot be closed since the entrance
to the tunnel is now widened (Figure S1).
In contrast to the significant effects in aldehyde channeling
when the residues at the entrance of the aldehyde tunnel are
replaced, replacement of Ile-195 at the exit of the tunnel in
BphJ with a larger phenylalanine did not reduce aldehyde
channeling. This implies that significant conformation changes
on the channel exit residues can occur to allow for aldehydes to
enter the dehydrogenase active site from the channel.
Activation of Aldolase Activity by the Dehydrogen-
ase. The asymmetric unit in the crystal of DmpG−DmpF
consists of two tetramers, with three of the four DmpF subunits
containing bound NAD+. In the crystal structure of DmpG−
DmpF, three residues at the exit of the dehydrogenase adopt
different conformations and only in the NAD+ bound form was
the channel exit opened for aldehyde to enter the dehydrogen-
ase active site. It has been determined that NADH activates the
aldolase reaction by 4-fold in the BphI−BphJ complex.4 The
BphI G322L, G323L, and G323F variants which completely
lack the ability to channel acetaldehyde were found to be
activated similar to the wild-type in the presence of NADH.
The activation of BphI by BphJ is due to allosteric
communication between the two enzymes rather than faster
aldehyde release through the channel.
Proposed Mechanism of Substrate Channeling. Based
on the results presented above, combined with previous work
on the catalytic mechanism of the aldolase,10 a mechanism for
aldehyde channeling in the aldolase−dehydrogenase complex is
proposed (Figure 5). In this mechanism the 4-hydroxy-2-
oxoacid enters the aldolase through the substrate tunnel where
it encounters His-20, which undergoes a conformation change
to allow the 4-hydroxy-2-oxoacid to enter the active site of the
aldolase. Following substrate entry, Tyr-290 rotates to
accommodate the substrate in the active site, thus opening
the aldehyde channel. The distal end of the alkyl side chains of
aldehydes >4C in length protrude into the channel. His-20
adopts a “closed” conformation which also enables it to abstract
a proton from C4-OH to initiate aldol cleavage. The aldehyde
is released into the channel allowing for the torsional
movement of Tyr 290 to a “closed” position, preventing the
aldehyde from diffusing back into the aldolase active site. His-
20 opens the substrate entry tunnel, allowing pyruvate to leave
and a second molecule of substrate to enter.
CONCLUSION
■
Analysis of the crystal structure of the aldolase−dehydrogenase
complex enabled the identification of a possible route for the
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direct channeling of aldehyde products from the aldolase to the
dehydrogenase. The channel appears to be dynamic, and
therefore the dimensions of the channel derived from the static
crystal structure are inadequate in predicting the sizes of
aldehydes that can diffuse through the channel. The
coordinated conformational changes of specific gating residues
appears to ensure the efficient channeling of aldehydes. The
results presented here complement our previous biochemical
data4,9,10 and provide a structural basis for understanding how
substrate channeling occurs in this enzyme complex.
ASSOCIATED CONTENT
■
S
* Supporting Information
Primers used in PCR site-directed mutagenesis (Table S1);
kinetic constants of the dehydrogenase associated with the
BphI glycine variants (Table S2); model of the L89A variant
(Figure S1). This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sseah@uoguelph.ca. Phone: (519) 824 4120 Ext
56750. Fax: (519) 837 1802.
Funding
This research is supported by National Science and Engineering
Research Council of Canada (NSERC), Grant 238284, to
S.Y.K.S. P.B. is a recipient of a NSERC PGS-D scholarship.
(16) Gouet, P., Courcelle, E., Stuart, D. I., and Metoz, F. (1999)
ESPript: analysis of multiple sequence alignments in PostScript.
Bioinformatics 15, 305−308.
ABBREVIATIONS
■
(17) Petrek, M., Kosinova, P., Koca, J., and Otyepka, M. (2007)
MOLE: a Voronoi diagram-based explorer of molecular channels,
pores, and tunnels. Structure 15, 1357−1363.
ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogen-
ase; HEPES, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic
acid; HOHA, 4-hydroxy-2-oxohexanoate; HOPA, 4-hydroxy-
2-oxopentanoate; IPTG, isopropyl β-D-thiogalactopyranoside;
LDH, L-lactate dehydrogenase.
(18) Delano, W. L. (2002) The PyMOL Molecular Graphics System,
DeLano Scientific, San Carlos, CA.
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quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248−254.
(20) Cornish-Bowden, A. (1995) Analysis of Enzyme Kinetic Data,
Oxford Univeristy Press, New York.
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