Download Expression and characterization of 1

Biochimica et Biophysica Acta 1703 (2004) 11 – 19
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Expression and characterization of 1-aminocyclopropane-1-carboxylate
deaminase from the rhizobacterium Pseudomonas putida UW4:
a key enzyme in bacterial plant growth promotion
Nikos Hontzeasa,1, Jérôme Zoidakisb, Bernard R. Glicka, Mahdi M. Abu-Omarc,*
a
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1
b
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA
c
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, USA
Received 18 June 2004; received in revised form 3 September 2004; accepted 8 September 2004
Available online 28 September 2004
Abstract
The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to aketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant
association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and
purified. It showed selectivity and high activity towards the substrate ACC: K M=3.4F0.2 mM and k cat=146F5 min-1 at pH 8.0 and 22 8C.
The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe
tapering in activity at higher pH resulting in loss of activity at pHN10. The major component of the enzyme’s secondary structure was
determined to be a-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 8C as determined by its CD temperature profile
as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this
mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to
the active site. This enzyme’s biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC
deaminases from other organisms can be compared.
D 2004 Elsevier B.V. All rights reserved.
Keywords: ACC deaminase; Pyridoxal 5-phosphate; Enzyme kinetics; Circular dichroism; Microcalorimetry
1. Introduction
The cylcopropanoid a-amino acid, 1-aminocyclopropane-1-carboxylate (ACC), is a precursor in the biosynthetic
pathway of the plant hormone ethylene, a simple gaseous
Abbreviations: AA, amino acid; ACC, 1-aminocyclopropane-1-carboxylate; ACCD, ACC deaminase; CD, circular dichroism; DSC, differential
scanning calorimetry; LDH, l-lactate dehydrogenase; MALDI-TOF,
matrix-assisted laser desorption/ionization-time of flight; PLP, pyridoxal
5-phosphate; PMP, pyridoxamine phosphate
* Corresponding author. Tel.: +1 765 494 5302; fax: +1 765 494 0239.
E-mail address: [email protected] (M.M. Abu-Omar).
1
Current address: Department of Chemistry, Purdue University, 560
Oval Drive, West Lafayette, Indiana 47907, USA.
1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2004.09.015
molecule that has been associated with many physiological
plant processes including senescence [1]. In higher plants,
S-adenosylmethionine is converted to ACC by the pyridoxal
5-phosphate (PLP)-dependent enzyme ACC synthase [2]. In
the final step of ethylene production, ACC is oxidized with
molecular oxygen by the action of the non-heme irondependent enzyme ACC oxidase to yield HCN and CO2 in
addition to ethylene [3]. Several strains of plant growthpromoting soil bacteria have been found to contain ACC
deaminase (ACCD), a PLP-dependent enzyme that converts
ACC to a-ketobutyrate and ammonium, Eq. (1) [4–6].
Introduction of ACCD in higher plants by gene modification
technology reduced the production of ethylene and delayed
ripening of fruits [7]. The number of ACC deaminases
recognized in the literature is limited. ACCD has been
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N. Hontzeas et al. / Biochimica et Biophysica Acta 1703 (2004) 11–19
isolated from a few strains of Pseudomonas species [5,8],
yeast Hansenula saturnus [9], and fungus Penicillium
citrinum [10].
2. Materials and methods
2.1. Bacterial strains
P. putida UW4 was isolated from the rhizosphere of
reeds growing on the campus of the University of Waterloo
based on its ability to utilize ACC as a sole source of
nitrogen and promote the growth of canola seedlings under
gnotobiotic conditions [6].
ð1Þ
PLP-dependent enzymes catalyze a wide variety of
biological reactions including transamination, racemization,
deamination, and decarboxylation. The PLP cofactor forms
a covalent Schiff base intermediate with the substrate
(referred to as the external aldimine) in place of an internal
Schiff base linkage (internal aldimine) with an active site
lysine residue. The accepted mechanism involves loss of a
proton from the external aldimine’s a carbon to form a
quinonoid intermediate [11]. Reprotonation yields a ketimine, which contains a double bond between the nitrogen
and a carbon of the substrate. The ketimine is finally
hydrolyzed to an a-keto acid and pyridoxamine phosphate
(PMP). Nevertheless, the cyclopropane ring-opening reaction of ACCD described in Eq. (1) is regarded as a special
case because its substrate does not contain a hydrogen atom
on the a carbon and the carboxyl group is retained in the
product.
A novel ACCD containing bacterium, Pseudomonas
putida UW4, has been shown to promote plant growth
under different environmental stresses including flooding
[12], drought [13], and the presence of heavy metals [14]
and phytopathogens [15]. The possibility of a close
mutualistic relationship between the plants and the soil
bacterium has been suggested, and the role of ACCD in
ensuring low levels of ethylene at critical stages of root
growth has been proposed [16]. Recent studies have
demonstrated that plants treated with ACCD containing P.
putida UW4 (as opposed to a knockout mutant) display
down-regulation of stress and defense genes but upregulation of growth-associated genes [17]. Despite the
biological studies on the effects P. putida UW4 has on plant
growth, little is known about the biochemical properties of
its ACCD and how this novel enzyme compares to other
characterized ACC deaminases. Herein, we report the
subcloning, expression, and kinetic and thermodynamic
characterization of P. putida UW4 ACCD. Our findings add
to the limited database of this enzyme’s biochemical and
biophysical properties and provide a valuable reference to
which newly isolated ACC deaminases can be compared.
We also present a novel point mutant in which Gly44, a
residue that is distant from the active site PLP cofactor, was
mutated to Asp44 yielding an inactive enzyme. By
comparison to the known structure of H. saturnus ACCD
[18], we suggest a previously unrecognized role for this
highly conserved residue.
2.2. Subcloning of the ACC deaminase gene
Two primers complimentary to the sequence at each end
of the ACC deaminase gene from P. putida UW4 were
prepared to amplify the gene from the pUC18 plasmid [19].
The forward primer 5V-GGGACCGGATCCTCAAGGAACAGCGCCATG-3Vcontains a BamHI site (in bold) and the
reverse primer 5V-GAACGGAAGCTTCTGGCGGCGCCAAGCTCA-3V contains a HindIII site downstream of the
stop codon (in bold). Primers were purchased from SigmaAldrich (Oakville, Ontario, Canada). The PCR amplified
DNA product was gel purified and digested with BamHI
and HindIII (Promega, Madison) and ligated to the BamHI/
HindIII sites of pET30a (Novagen, Madison). The resulting
construct pNH01 was subsequently used to transform E.
coli BL21(DE3)pLysS (Novagen). Methods and protocols
for recombinant DNA manipulations were carried out
according to the manufacturers’ manuals or from general
references [20].
2.3. Expression and purification of recombinant ACC
deaminase
An overnight culture of E. coli BL21(DE3)pLysS/
pNH01 was grown in 1 L of LB supplemented with
kanamycin (30 Ag/mL; Promega) and chloramphenicol (34
Ag/mL; Promega) at 37 8C. When the OD600 reached 0.4,
the culture was induced with IPTG (Promega) to a final
concentration of 1 mM and allowed to grow for an
additional 3 h at 37 8C. The cells were harvested by
centrifugation at 8000g for 10 min at 4 8C and then the
bacteria were lysed using BugBuster (Novagen). Cell debris
was removed by ultra-centrifugation at 30 000g for 45
min. The supernatant was then poured in a column
containing His-bind resin (Novagen) and the recombinant
ACC deaminase was purified according to the manufacturer’s instructions. Briefly, 4 mL of His-tag resin was loaded
into a column and charged with 50 mM NiSO4 and
equilibrated with 0.5 M NaCl, 20 mM Tris–HCl, 5 mM
imidazole (pH 7.9). The crude ACCD supernatant was then
poured through the column and the column was washed
with 0.5 M NaCl, 60 mM imidazole, 20 mM Tris–HCl (pH
7.9). Purified ACCD was eluted with 1 M imidazole, 0.5 M
NaCl, 20 mM Tris–HCl (pH 7.9). The purified ACCD was
dialyzed at room temperature and the buffer was exchanged
with 0.1 M phosphate buffer (pH 8.0). The buffer
N. Hontzeas et al. / Biochimica et Biophysica Acta 1703 (2004) 11–19
exchanged purified enzyme was used for subsequent
experiments. The overall molecular weight of the purified
recombinant ACCD was measured by matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass
spectrometry to an accuracy of F0.02% at the Pasarow
Mass Spectrometry Laboratory at UCLA.
2.4. Enzyme assays
UV–Vis spectra and steady-state kinetics were recorded
on a Shimadzu UV-2501 double-beam spectrophotometer
equipped with a thermostat cell holder. Enzyme activity was
measured by a coupled assay with l-lactate dehydrogenase
(LDH; Sigma), following the disappearance of NADH
(Sigma) at 340 nm (e=6220 M1 cm1). A standard assay
was performed in 100 mM phosphate buffer (pH 8.0) at 22
8C, containing 40 mM ACC, 1 AM ACCD, 0.30 mM
NADH and 30 U/mL LDH. In order to determine the K M the
ACC concentration was varied between 0.50 and 40 mM.
Data were fitted to the Michaelis–Menten equation using the
program KaleidaGraph 3.1. The buffers used for the pH
dependence studies were phosphate (5.8–8.8), Tris (8.4–9.2)
and Glycine (9.2–10.6). For the temperature dependence
studies, the range was from 10 to 37 8C. The activation
parameters were calculated as previously described [21].
2.5. Random mutagenesis of the ACC deaminase gene
The ACC deaminase gene from P. putida UW4 was used
as a template to perform PCR-based random mutagenesis.
To perform error-prone PCR, a pGEM-Teasy vector construct was used, into which the ACCD gene from the
bacterium was inserted within the multiple cloning site of
the vector. This served as template DNA and was diluted to
1 ng/AL. To achieve 1–2 mutations per 1000 base pairs, the
following PCR reaction was setup: 40-AL deionized H2O, 5AL 10 Taq buffer (Fermentas), 1 AL of 2 mM dGTP,
dNTPs (Fermentas), 10 AM each primer (sense direction: 5VGGGACCGGATCCTCAAGGAACAGCGCCATG-3V and
antisense direction: 5V-GAACGGAAGCTTCTGGCGGCGCCAAGCTCA-3V), 1 ng/AL template DNA and 1-AL Taq
polymerase (Fermentas). PCR was performed in a MJ PTC100 thermocycler using the following conditions: 94 8C for
30-s denaturation, 25 cycles of 94 8C denaturation for 30 s
and 68 8C extension for 1 min followed by a final step of 72
8C for 1 min. The PCR product obtained was visualized on a
1% agarose gel stained with ethidium bromide and a band
approximately 1 kb was gel purified using the Montage
DNA purification system (Amicon).
2.6. Cloning and screening for mutant ACC deaminase
constructs
Following error-prone PCR, a bpoolQ of potential mutant
PCR DNA products was obtained with a size of 1 kb. The
DNA was ligated into the pGEM-Teasy vector system and
13
E. coli JM109 competent cells were transformed with the
vector. The bacteria were plated on LB plates with 100 Ag/
mL of ampicillin and then incubated at 37 8C for 16 h.
Colonies from the plates were then pooled with 1 mL of
0.85% saline and a 5-mL LB overnight culture with 100 Ag/
mL ampicillin was inoculated with 5 AL of the pooled
colonies grown at 37 8C, 200 rpm shaking overnight.
Following this, plasmid DNA was isolated from the overnight culture [20].
2.7. Double digestion and transformation into expression
host cells
The bpoolQ of plasmid DNA obtained from above was
digested with both BamHI and HindIII. Specifically, a 100AL reaction was set up containing 10 AL of the appropriate
restriction digest buffer according to the manufacturer’s
instructions (Promega), 5 AL each of BamHI and HindIII,
10 AL of plasmid DNA and 70 AL of distilled water. The
reaction was carried at 37 8C overnight, after which the
restriction enzymes were inactivated by incubating at 85 8C
for 30 min. The sample was then dried in a Savant DNA110
Speedvac, and resuspended in 10 AL of sterile distilled
water. The sample was subsequently run on a 1% agarose
gel and a band corresponding to 1 kb (pooled potential ACC
deaminase mutated genes) was excised and gel purified
using the Montage DNA purification system according to
the manufacturer’s instructions. After measuring the volume
of the sample eluted from the Montage columns, one-tenth
the volume of sodium acetate pH 5.5 was added, and to the
combined volume 1.5 volumes of 100% ethanol was added.
This mixture was incubated at 80 8C for 30 min and then
centrifuged in an Eppendorf 5810R refrigerated centrifuge
for 10 min at maximum speed. The pellet obtained was
washed twice with 70% ethanol and then the pellet was
dried in a Savant DNA110 Speedvac, and resuspended in 10
AL of sterile distilled water. The DNA concentration of the
sample was determined spectrophotometrically at 260 nm.
The digested DNA was then ligated to the pET-30a vector
(Novagen) which had been previously digested with BamHI
and HindIII and purified the same way as the bpoolQ of ACC
deaminase genes described above. Ligation was carried out
using a 3:1 insert to vector molar ratio. In a 10-AL volume,
there was 5 AL of ligation buffer (Promega), 1-AL ligase
(Promega), appropriate amount of digested pET-30a
vector and digested ACC-deaminase, and distilled water
up to a volume of 10 AL. The reaction was carried
overnight at 4 8C. The ligation product was used to
transform expression host cells E. coli BL21(DE3)pLysS
and the cells were plated on LB plates containing 34-AL
chloramphenicol and 30-AL kanamycin. E. coli
BL21(DE3)pLysS cells contain a chloramphenicol resistance gene while the pET vector contains a kanamycin
resistance gene. The plates were incubated at 37 8C for
14–16 h. A control was included with wild-type ACC
deaminase from P. putida.
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2.8. Screening for mutated ACC deaminase
After overnight growth, colonies appearing on LB
plates were pooled using 1 mL of 0.85% saline,
vigorously vortexed, and washed three times with 4-mL
0.85% saline. After the washes, the pellet was resuspended in 1-mL 0.85 % saline. The solution was then
streaked onto LB plates containing 34-AL chloramphenicol, 30-AL kanamycin, 0.4 mM IPTG (Promega) and 5
mM ACC (Calbiochem), and incubated overnight at 37
8C. After incubation the plates were replica-plated onto
M9 plates also containing 34-AL chloramphenicol, 30-AL
kanamycin, 0.4 mM IPTG (Promega) and 5 mM ACC.
Colonies that did not grow after replica plating were
selected, and the insert was sequenced.
2.9. Site-directed mutagenesis of ACC deaminase gene
UW4 ACC deaminase gene insert, pure recombinant
ACCD was obtained by purification of crude bacterial
extracts through a nickel-column. A typical Coomassie
blue-stained gel from SDS-PAGE of purified ACCD is
shown in Fig. 1A along with its estimated molecular
weight of approximately 42 kDa. MALDI-TOF mass
spectral analysis revealed a mass to charge ratio of
41,848 Da. This value is in agreement with the calculated
molar mass of the translated gene sequence (41,842 Da).
Other ACC deaminases purified from the yeast H.
saturnus [9] and the fungus P. citrinum [10] have a
molar mass in the range of 40 to 41 kDa. The UV–Vis
spectrum of purified recombinant ACCD (Fig. 1B)
displays characteristics typical of PLP-dependent enzymes
(A 328/A 418~2.5). The absorption maxima at 418 and 328
nm are indicative of the protonated internal PLP-aldimine
tautomeric forms ketoenamine and enolimine, respectively
[23].
Site-directed mutagenesis was carried out using the
QuickchangeII XL-site directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.
2.10. Circular dichroism (CD)
CD studies were performed with a Jasco J-710 spectrophotometer equipped with a Peltier temperature controller. The CD spectra were measured from 196 to 260 nm
using a 1.00-mm quartz cell. The protein concentration was
5.0 AM in 100 mM phosphate (pH 8.0). The mean residue
molar ellipticity for the recombinant ACCD (393 a.a.) was
calculated using a molecular weight of 41,820 Da. The
secondary structure estimation from CD spectra was
performed with the program Softsec 1.2 [22]. The thermal
unfolding of ACCD was monitored by measuring the
ellipticity at 222 nm. The protein concentration was 11
AM and the temperature was varied from 35 to 85 8C with a
scan rate of 1 8C/min. The inflection point of the ellipticity
vs. temperature plot yielded the melting temperature of the
enzyme (T m).
2.11. Differential scanning calorimetry (DSC)
DSC studies were performed with Calorimetry Sciences
Corporation N-DSC II differential scanning microcalorimeter. The protein concentration was 37.5 AM in 100 mM
phosphate (pH 8.0). The scan rate was 1 8C/min. The data
were analyzed using the program CpCalc Analysis 2.1.
3. Results
3.1. Gene cloning, purification and characterization of
P. putida ACCD
After induction with IPTG of E. coli BL21(DE3)pLysS containing the pET 30a vector with the P. putida
Fig. 1. (A) SDS-PAGE of crude and purified P. putida UW4 ACC
deaminase. (B) UV–Vis spectrum of recombinant ACCD.
N. Hontzeas et al. / Biochimica et Biophysica Acta 1703 (2004) 11–19
15
3.2. Kinetic characterization, activation parameters, and
pH profile of ACCD
Catalytic velocities of recombinant ACCD were measured at varying substrate concentrations (Fig. 2A). Data
fitting to the Michaelis–Menten equation yield for ACC a
K M=3.4F0.2 mM and a k cat=146F5 min1 at pH 8.0 and
22 8C. The K M value determined for P. putida UW4 ACCD
is in the range (2 to 17 mM) previously observed for crude
enzyme extracts of 12 different ACCD-containing microorganisms [7,24,25]. The overall catalytic efficiency as
indicated by the second order rate constant k cat/K M=716
M1 s1 of our UW4 ACCD is comparable to that reported
by others for Pseudomonas ACCD, 690 M1 s1 [26]. In
addition to ACC, d-serine as well as other ACC-related
substrates such as di-coronamic acid and dimethyl-ACC is
an active substrate for the enzyme. However, the enzyme
does not recognize l-amino acids.
The effect of pH on ACCD activity was investigated
over a wide range (Fig. 2B). The data fit best to a bell-
Fig. 3. (A) CD spectrum of wild-type ACCD in its native form. (B) Melting
curve for ACCD as determined by CD following the a-helical secondary
structure at 222 nm.
shaped curve with minima of 0 at both pH extremes. The
enzyme showed highest activity at pH 8.0 and the k cat
values decreased dramatically below pH 7.5 and above
pH 9.0. The pH dependence yielded pK a values of 7.4
and 9.5 (Fig. 2B).
In the limit of substrate (ACC) saturation, we determined
enzymatic activity at different temperatures in the range of
10 to 37 8C. The temperature profile of the enzyme (ln k cat
versus 1/T) yielded the following activation parameters:
DH z=47F2 kJ mol1 and DS z=78F5 J mol1 K1. These
values give a DG z (298 K)=70F1 kJ mol1.
3.3. Circular dichroism (CD), secondary structure, and
thermal stability of ACCD
Fig. 2. Kinetic dependence on substrate (ACC) concentration fitted to the
Michaelis–Menten equation. (B) pH profile for P. putida UW4 ACCD.
In order to probe the secondary structure of P. putida
UW4 ACCD, its CD spectrum was acquired (Fig. 3A)
and analyzed using the self-consistent singular decomposition algorithm developed by Sreerama and Woody
[22]. The main feature of the CD spectrum is the strong
signal at 222 nm that is characteristic for a helical
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N. Hontzeas et al. / Biochimica et Biophysica Acta 1703 (2004) 11–19
proteins. The deconvolution of the CD spectrum gave the
following secondary structural components for P. putida
UW4 ACCD: 30% a helix, 9% h strand, 21% h turn,
and 40% unclassified coil structures.
The thermodynamic stability of recombinant P. putida
UW4 ACCD was investigated by following the enzyme’s
a helical secondary structure in the CD at 222 nm as a
function of temperature. The denatured protein lacked a
CD signal indicative of loss of secondary structure. The
protein’s melting temperature was determined to be 60F2
8C from plots of molar ellipticity versus temperature (Fig.
3B) [27]. Following denaturation, cooling ACCD back to
ambient temperature did not restore its secondary
structure. Therefore, thermal unfolding of ACCD was
irreversible.
The enzyme’s melting temperature was also confirmed
by differential scanning microcalorimetry (DSC). A typical
plot of the specific heat capacity at constant pressure (C P)
versus temperature is displayed in Fig. 4. The melting
temperature by DSC was determined to be 58F1 8C. The
sloping in the baseline prior to denaturation is indicative
of water loss from the protein preceding the unfolding
transition.
3.4. The G44D point mutant
In screening for single point mutants involving
residues that are distant from the active site PLP cofactor
but highly conserved in known ACCD sequences, we
discovered that the mutant G44D P. putida UW4 ACCD
is totally inactive. Back mutation of residue Asp44 to
Gly by site-directed mutagenesis yielded a fully active
enzyme. Furthermore, the CD spectrum of the mutant
(data not shown) is identical to that of the wild-type
enzyme. In addition, plots of molar ellipticity at 222 nm
versus temperature (data not shown) for the G44D
mutant enzyme yielded a melting temperature of 60 8C,
Fig. 4. DSC melting profile for ACCD.
which is indistinguishable from that obtained for the
native enzyme.
4. Discussion
While ACCD has been suggested to play a key role in
free-living plant growth-promoting bacteria [16], the
number of isolated and characterized enzymes in the
literature remains limited [8–10]. Secondly, ACCD has
generated much interest in recent years due to the belief
that a novel mechanism is operative in its cyclopropane
ring-opening reaction [26,28]. The substrate, ACC, lacks
an a hydrogen, and thus a focal point of these studies
has been the enzyme’s reaction mechanism. Two plausible pathways have been suggested [29]: (1) nucleophilic
addition to open the ring followed by h-proton abstraction; (2) direct h-proton abstraction to initiate cyclopropane cleavage.
We have prepared and characterized a novel ACCD
from P. putida UW4. The translated amino acid sequence
of this enzyme is compared in Fig. 5 with other
characterized ACC deaminases from different species.
The amino acid (AA) sequence identity between P. putida
UW4 ACCD and the yeast enzyme is quite good (60%).
The key residues associated with the active site PLP
cofactor (Tyr268, Tyr294, Lys51, and Glu295, P. putida
UW4 ACCD numbering scheme) are all conserved. The
crystal structure of H. saturnus ACCD has been solved
[18], and it provides a guide for comparison. Despite the
reasonable AA identity of our ACCD with that of the
yeast, the secondary structure components of both enzymes
differ (Table 1). It is worth noting, however, that the most
reliable secondary structural element deduced from CD
spectra is a helix, which remains sufficiently different in
the two enzymes.
Enzymatic activity was investigated through a
coupled assay with LDH, Eq. (2). The kinetics of NADH
disappearance at 340 nm featured zero-order dependence on
[NADH], which is consistent with the ACCD reaction being
rate determining in Eq. (2). Furthermore, the rate of reaction
displayed first-order dependence on the enzyme ACCD
concentration over a broad range. The catalytic activity (K M
and k cat) of our recombinant enzyme compares well with
other ACC deaminases from different microorganisms
[7,24,25]. With respect to k cat, P. putida UW4 ACCD is
among the most active; as for K M, it displays one of the
lowest values reported, the range for other ACC deaminases
being 2 to 17 mM. Poor substrate binding, i.e., a K M in the
millimolar range, is a recognized characteristic of ACC
deaminases. For biotechnological applications, improving
upon ACCD efficiency (defined as k cat/K M) could be
achieved by increasing k cat or, conversely, decreasing K M.
It is the latter in this instance that needs improvement.
Rational design or random mutagenesis to yield a more
efficient ACCD is a worthy goal. The activation parameters
N. Hontzeas et al. / Biochimica et Biophysica Acta 1703 (2004) 11–19
17
Fig. 5. Alignments of amino acid sequences for ACC deaminases. Asterisks designate the amino acid residues that are conserved in all five sequences. Dashes
indicate gaps inserted to optimize the alignment. Glycine 44 and Lysine 51 are indicated in bold as well as P. putida UW4.
for k cat feature a typical enthalpy of activation and large
negative entropy of activation. These values are consistent with a covalent association of the substrate with the
enzyme and a highly organized transition state.
ð2Þ
Optimal activity for P. putida UW4 ACCD was observed
at pH 8 with total loss of activity below pH 7. The decline in
activity at pH values higher than 8 is somewhat more
gradual resulting in total loss of activity above pH 10 (Fig.
2B). Recent mutagenesis and structural studies of yeast
ACCD [30] have pinpointed the involvement of the
following active site residues in catalysis: Tyr269, Tyr295,
Lys51, and Glu296 (yeast ACCD numbering sequence). The
same study has also suggested a novel role for Lys51 as the
residue responsible for proton abstraction from the substrate
(ACC) with assistance from other residues, namely, Tyr295
and 269. In the pH range investigated, the internal PLPaldimine remains protonated and the dipolar aldimine is not
present at any notable concentrations below pH 11 [31].
Therefore, the observed pK a of ~7.4, which controls k cat, is
not deprotonation of the aldimine nitrogen. Instead, it must
involve an active site residue whose protonation state affects
the ratio between ketoenamine and enolimine tautomers.
Similar observations have been noted for the pyridoxal
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Table 1
Comparison of secondary structure for ACCD from P. putida UW4 and H. saturnus
ACCD
a Helix (%)
h Strand(%)
h Turn (%)
Unclassified coil structures (%)
Reference
P. putida
H. saturnus
30
42.8
9
15.8
21
21.4
40
20.8
This work
[18]
phosphate-dependent dialkylglycine decarboxylase [31]. In
light of the structural results on the yeast ACCD [30] and
our pH dependence, it is conceivable that the pK a of Lys51
(P. putida UW4 ACCD numbering) is modulated in the
protein’s hydrophobic environment of the active site by
almost three orders of magnitude (from a pK a of 10 in free
solution to 7.5 in the enzyme) [32]. In the higher pH limit,
the observed pK a of 9.5 can be attributed to the ionization of
active-site residues Tyr268 and Tyr294 (P. putida UW4
ACCD numbering). Their hydrogen bonds to the internal
aldimine, which has been suggested in activating the
substrate ACC towards deprotonation [30], would be
disrupted.
P. putida UW4 ACCD proved to be a thermodynamically
stable enzyme as evidenced by its melting temperature. It is
interesting to note that based on the amino acid sequence,
the software program bProtParamQ tool (available at the
ExPASy web site at www.expasy.org) predicted an unstable
protein. In comparison, ACC synthase as well as ACC
oxidase has a short half-life and exists in low abundance in
most plant tissues.
While screening for highly conserved residues that are
distant from the active site, we discovered the point mutant
G44D P. putida UW4 ACCD, which corresponds to the
yeast Gly44. This mutant behaves similarly to the native
enzyme in terms of expression, handling, secondary
structure, UV–Vis, and thermodynamic stability. However,
it displays no activity whatsoever even at ACC concentrations higher than 40 mM. Using the structure for yeast
ACCD, we modeled Asp44 in place of Gly using the
program SPDBV found at the ExPASy web site, www.
expasy.org. The negatively charged Asp in position 44
protrudes into the solvent region, locking the loop D39-N50
into a position that blocks substrate access to the buried
active site. Furthermore, when the residue Asp44 is mutated
back into the original Gly, the enzyme’s full activity is
restored. Therefore, we conclude that the highly conserved
residue Gly44 is important in gating ACC (substrate) entry
to the enzyme’s active site.
In conclusion, P. putida UW4 ACC deaminase is readily
expressed in E. coli in good yields. It is a robust enzyme
with activity towards ACC that is among the best reported in
the literature. Glycine 44 has been tentatively identified as a
key residue in allowing a pathway for substrate access to the
active site. For biotechnological applications, an ACCD
with improved substrate affinity would be desirable. In
working towards this goal, we are currently isolating and
characterizing ACC deaminase proteins from different
bacterial species.
Acknowledgments
Nikos Hontzeas is a recipient of a National Sciences and
Engineering Research Council of Canada (NSERC) postgraduate scholarship. Financial support for this research was
provided by NSERC (to B.R.G) and NSF (to M.M.A.-O.).
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