Indeed, the valine rotamer, which better fits the C2,3O active site, has one of the methyl groups at approximately the same position as the Thr249 methyl group that approaches the substra
Trang 1catalytic efficiency and substrate specificity of
catechol-2,3-dioxygenase from Pseudomonas stutzeri OX1 Loredana Siani1,*, Ambra Viggiani1,*, Eugenio Notomista1, Alessandro Pezzella2
and Alberto Di Donato1
1 Dipartimento di Biologia Strutturale e Funzionale, Universita` di Napoli Federico II, Napoli and CEINGE-Biotecnologie Avanzate S.c.ar.l., Italy
2 Dipartimento di Chimica Organica e Biochimica, Universita` di Napoli Federico II, Italy
Several bacteria are capable of using aromatic
hydro-carbons as growth substrates [1–4] The remarkable
range of substrates that can be metabolized endows
these microorganisms with the potential for
bioremedi-ating environmentally dangerous substances such as
benzene, toluene, xylene isomers, and polycyclic aro-matic hydrocarbons and their derivatives [5–8] Because of their toxicity, several of these compounds are in the US Environmental Protection Agency prior-ity pollutant list (http://www.epa.gov) For example
Keywords
bioremediation; dioxygenase; enzyme
kinetics; protein expression; Pseudomonas
stutzeri
Correspondence
A Di Donato, Dipartimento di Biologia
Strutturale e Funzionale, Universita` di Napoli
Federico II, Via Cinthia, I-80126 Napoli, Italy
Fax: +39 081 676710
Tel: +39 081 679143
E-mail: didonato@unina.it
*These authors contributed equally to this
work
(Received 27 January 2006, revised
28 March 2006, accepted 4 May 2006)
doi:10.1111/j.1742-4658.2006.05307.x
Bioremediation strategies use microorganisms to remove hazardous sub-stances, such as aromatic molecules, from polluted sites The applicability
of these techniques would greatly benefit from the expansion of the cata-bolic ability of these bacteria in transforming a variety of aromatic com-pounds Catechol-2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1
is a key enzyme in the catabolic pathway for aromatic molecules Its specif-icity and regioselectivity control the range of molecules degraded through the catabolic pathway of the microorganism that is able to use aromatic hydrocarbons as growth substrates We have used in silico substrate dock-ing procedures to investigate the molecular determinants that direct the enzyme substrate specificity In particular, we looked for a possible molecular explanation of the inability of catechol-2,3-dioxygenase to cleave 3,5-dimethylcatechol and 3,6-dimethylcatechol and of the efficient clea-vage of 3,4-dimethylcatechol The docking study suggested that reduction
in the volume of the side chain of residue 249 could allow the binding of 3,5-dimethylcatechol and 3,6-dimethylcatechol This information was used
to prepare and characterize mutants at position 249 The kinetic and regio-specificity parameters of the mutants confirm the docking predictions, and indicate that this position controls the substrate specificity of catechol-2,3-dioxygenase Moreover, our results suggest that Thr249 also plays a previ-ously unsuspected role in the catalytic mechanism of substrate cleavage The hypothesis is advanced that a water molecule bound between one of the hydroxyl groups of the substrate and the side chain of Thr249 favors the deprotonation⁄ protonation of this hydroxyl group, thus assisting the final steps of the cleavage reaction
Abbreviations
C2,3O, catechol-2,3-dioxygenase; DHBD, 2,3-dihydroxybiphenyl-1,2-dioxygenase; DHND, 1,2-dihydroxynaphthalene dioxygenase; DHpCD, 2,3-dihydroxy-p-cumate dioxygenase; DMC, dimethylcatechol; ECD, extradiol ring cleavage dioxygenase; HPCD, 3,4-dihydroxyphenylacetate (homoprotocatechuate)-2,3-dioxygenase; IBX, o-iodoxybenzoic acid; 3-MC, 3-methylcatechol; 4-MC, 4-methylcatechol; PH, phenol
hydroxylase; THTD, 2,4,5-trihydroxytoluene-5,6-dioxygenase; ToMo, toluene ⁄ o-xylene monooxygenase.
Trang 2long-term exposure of humans to benzene, toluene and
xylene could cause damage to the central nervous
sys-tem, liver and kidneys, chromosomal aberrations and
cancer [9–12]
Extradiol ring cleavage dioxygenases (ECDs) are
Fe(II)-dependent enzymes that catalyze a crucial
ring-opening step in the catabolic pathways of
microorgan-isms capable of growing on aromatic compounds
[13–15] ECDs cleave ortho-dihydroxylated aromatic
rings by catalyzing the addition of two atoms from
molecular oxygen at one of the C–C bonds adjacent to
the diol (metacleavage; Fig 1) to produce nonaromatic
molecules that eventually enter central metabolic
path-ways [13,14] ECDs comprise five evolutionarily related
subfamilies [16] that include catechol-2,3-dioxygenase
(C2,3O; EC 1.13.11.2) (subfamily 1),
2,3-dihydroxybi-phenyl-1,2-dioxygenase (DHBD; EC 1.13.11.39) and
1,2-dihydroxynaphthalene dioxygenase (DHND,
sub-family 2), 3,4-dihydroxyphenylacetate-2,3-dioxygenase
(HPCD; EC 1.13.11.15) (subfamily 3),
2,3-dihydroxy-p-cumate-3,4-dioxygenase (DHpCD, subfamily 5) and
2,4,5-trihydroxytoluene-5,6-dioxygenase (THTD,
sub-family 6) Even though the range of substrates that
can be oxidized by ECDs is broad, each enzyme in the
family displays restricted substrate specificity and reg-ioselectivity ECDs belonging to subfamilies 1, 2 and 3 cleave catechols substituted at positions 3 and⁄ or 4 at the bond adjacent to the diol and proximal to the sub-stituent, as shown in Fig 1A [17–23] ECDs belonging
to subfamilies 5 and 6, such as DHpCD and THTD, catalyze the transformation of 3,6-disubstituted and 4,5-disubstituted catechols, respectively [24–26], and exhibit high regioselectivity by cleaving the bond prox-imal to the alkylic group of the substrates as shown in Fig 1B,C [24–26] The size of the substitutent that can
be accommodated by a subfamily varies For instance, C2,3Os can cleave catechols with small substituents at positions 3 and 4, such as 3-methylcatechol (3-MC) and 3,4-dimethylcatechol (3,4-DMC) [17,18], whereas enzymes belonging to subfamily 2 act on catechols with large substituents at the same positions [19–21,27] (Fig 1A)
The complete degradation of aromatic molecules is initiated by monooxygenases and dioxygenases, which produce dihydroxylated compounds in the upper metabolic pathways [28,29] These diols are cleaved subsequently by ECDs Since monooxygenases and dioxygenases usually exhibit a wide range of substrate specificity, they produce several dihydroxylated prod-ucts, some of which are not always substrates for ECDs and cannot be degraded further As a conse-quence, ECDs represent the gate that controls the flow
of molecules entering the lower metabolic pathways [14,28,29], by reducing the range of aromatic com-pounds that can be used by microorganisms as growth substrates Thus, enhancement of the catabolic poten-tial of ECDs would represent a valuable tool for bio-remediation strategies by widening the number of substrates that can be consumed by bacteria that depend on these enzymes for the utilization of specific aromatic substrates as their primary source of carbon and energy
Pseudomonas stutzeri OX1 is an ideal model organ-ism for these studies, since it can utilize benzene, tolu-ene, and o-xyltolu-ene, but not m-xylene and p-xyltolu-ene, as sole sources of carbon and energy [30] Two NADH-dependent monooxygenases—toluene⁄ o-xylene mono-oxygenase (ToMO) and phenol hydroxylase (PH)—act sequentially in the microorganism [31] to convert aro-matic hydrocarbons to the corresponding catechols These are cleaved by a C2,3O that is nearly identical
to the well-characterized enzyme from Pseudomonas putidaMT2 [18,32] ToMO and PH are able to convert o-xylene as well as m-xylene and p-xylene to 3,4-DMC, 3,5-DMC and 3,6-DMC, respectively (unpublished results) However, P stutzeri C2,3O can cleave only 3,4-DMC effectively [32], allowing this product to be
Fig 1 Scheme of the reactions catalyzed by extradiol ring cleavage
dioxygenases (ECDs) Reactions catalyzed by (A)
catechol-2,3-dioxy-genases (C2,3Os), 2,3-dihydroxybiphenyl-1,2-dioxycatechol-2,3-dioxy-genases (DHBDs)
and 3,4-dihydroxyphenylactetate-2,3-dioxygenase (HPCD), (B) by
2,3-dihydroxy-p-cumate dioxygenases (DHpCDs), and (C) by
2,4,5-trihydroxytoluene-5,6,dioxygenases (THTDs).
Trang 3further metabolized through the lower pathway This
is not possible in the case of 3,5-DMC and 3,6-DMC,
because of the very low activity of C2,3O towards
these compounds [32] Thus, the restricted specificity
of C2,3O is the primary metabolic determinant that
limits the ability of P stutzeri OX1 to efficiently grow
on xylene mixtures Moreover, the inability of P
stut-zeriC2,3O to cleave 3,5-DMC and 3,6-DMC also has
an adverse effect on the metabolism of the
micro-organism, since the NADH consumed by the
mono-oxygenase-catalyzed hydroxylations of m-xylene and
p-xylene cannot be restored by the lower pathway
reac-tions This inefficiency results in a loss of metabolic
reducing power when P stutzeri OX1 grows on xylene
mixtures An understanding of the molecular
determi-nants that control the substrate specificity of P stutzeri
C2,3O offers an opportunity to develop molecular
strategies aimed at adjusting the active site pocket of
C2,3O to control the products of the enzyme-catalyzed
reaction Such adjustment could enhance the ability of
the microorganism to grow on substituted aromatic
compounds Here, we report a study of the molecular
determinants of C2,3O substrate specificity carried out
by in silico substrate docking procedures followed by
the preparation and characterization of mutants at
position 249 Our findings indicate that Thr249
partici-pates in the control of substrate specificity and plays a
previously unsuspected role in catalysis
Results
Modeling of (di)methylcatechols in the active site
of C2,3O
C2,3Os from P putida MT2 and P stutzeri OX1 have
nearly identical C-terminal catalytic domains, except
for a single conservative substitution of leucine for
valine at position 225 in the P stutzeri enzyme Since
this substitution is 14 A˚ from the catalytic iron atom,
it is likely that the active sites of the two C2,3Os are
structurally identical and that the crystal structure of
P putida MT2 C2,3O (PDB accession code, 1mpy
[33]) would serve as an accurate model for
investi-gating the interactions of docked methylcatechols and
dimethylcatechols with the C2,3O substrate-binding
The structures of two ECDs, DHBD from
Pseudo-monasKKS102 (1eim [34]), and HPCD from
Brevibac-terium fuscum (1q0c [35]), crystallized in their active
Fe(II) form with the substrate bound to the catalytic
metal, were used as templates for initial positioning of
catechols in the active site of C2,3O The available
data suggest that the two structures (Fig 2A,B)
repre-sent the catalytically competent enzyme–substrate com-plex [34,35] First, the catalytic C2,3O iron atom and three ligands (His154, His214, Glu265) were superim-posed on the corresponding atoms of DHBD (His145, His209, Glu260) and HPCD (His155, His214, Glu267) After superimposition of the active site atoms of C2,3O on the corresponding atoms of DHBD and HPCD, r.m.s.d values were 0.35 A˚ and 0.24 A˚, res-pectively Then, a (substituted) catechol molecule was superimposed on the corresponding atoms of dihydroxy-biphenylacetate or dihydroxyphenylacetate to obtain two models of a catechol–C2,3O complex, named 1 and 2, respectively, in which the geometric parameters
of the metal center atoms are very similar to those found in the DHBD and HPCD structures The two models were inspected to find close molecular contacts between the catechol ring and the residues surrounding the binding pocket The two complexes were very sim-ilar In both structures, the largest contacts were found between the plane of the substrate ring and the plane
of the imidazole ring of residue His246, which make p contacts However, it should be noted that in complex
2, based on the HPCD structure, the average distance between the two interacting rings (3.0 A˚) is lower than that measured in complex 1 (3.6 A˚) The same distance
is 3.6 A˚ in the DHBD complex and 3.5 A˚ in the HPCD complex (Fig 2A,B) No other close molecular contacts were found in the two models Given the high similarity between the two complex models, complex 1, based on the DHBD structure, was selected for further analyses
Owing to changes in the conformation of the back-bone structures in C2,3O, the side chain of His246 is shifted towards the substrate, resulting in a larger overlap between the stacked rings Moreover, the side
of the substrate ring opposite to His246 faces the edge
of the Phe191 side chain (Phe186 in DHBD, Trp192 in HPCD) (not shown) The contacts between the edge of the dihydroxylated substrate ring and the active site pocket are probably involved in the determination of substrate specificity Inspection of the substrate CH atoms at positions 3 and 4 reveals that they point towards small cavities, indicated as subsites 1¢ and 2¢
in Fig 2C, which are defined by residues Ile204, Phe302, Ile291 and Leu248 Although the volume of subsite 2¢ is smaller than that of subsite 1¢, these cavit-ies are large enough to accommodate methyl substitu-ents at positions 3 and 4, as verified by the docking of 3-MC, 4-MC and 3,4-DMC A model of the complex between C2,3O and 3,4-DMC is depicted in Fig 2C
In HPCD, in contrast to what is observed in the model
of the C2,3O complex, the cavity corresponding to subsite 2¢ is larger and contains two arginine residues
Trang 4that interact with the carboxylate group of
homoproto-catechuate (Figs 1A and 2B) In DHBD, this region is
open to the solvent, thus allowing for the binding of
larger substituents (Fig 2A)
The CH atoms of the substrate ring at positions 5
and 6 point towards the backbone of Leu248 and the
side chain of Thr249, respectively (Fig 2C)
Appar-ently, the close contacts between these two residues
and the edge of the substrate ring could prevent
bind-ing of 3,6-DMC and 3,5-DMC, as shown in Fig
2-D,E,F Thus, the binding of 3,5-DMC and 3,6-DMC
to the active site of C2,3O could be possible if the
con-formation of the active site changes with respect to the
one observed in the crystal structure of C2,3O upon
binding of the dimethylcatechols
Since the CH atoms at position 5 of the substrate
ring point towards the backbone carbonyl group of
Leu248, replacement of the side chain at this position
would not be able to create space for accommodating
a methyl group at position 5 (Fig 2D,E,F) The CH atoms at position 6, however, contact the side chain of residue Thr249 The tightest substrate–enzyme contacts were located between the CH at position 6 and the methyl group of the Thr249 side chain In the four protomers of C2,3O, the Thr249 side chain shows the same orientation, probably due to a hydrogen bond between the OH group of Thr249 and the oxygen atom of the Leu248 carbonyl group (the two oxygen atoms are at 2.7 A˚ distance) A 180 rotation along the Ca–Cb bond would minimize the interaction between the side chain and the substrate bound in the putative productive conformation However, it would also prevent formation of the hydrogen bond between the Thr249 side chain and the backbone A reduction
in the volume of this side chain might provide room for housing a methyl substituent at this position and allow for the binding of 3,6-DMC or 3,5-DMC, as depicted in Fig 2D,E
Fig 2 Scheme of the active sites of 2,3-dihydroxybiphenyl-1,2-dioxygenase (DHBD), 3,4-dihydroxyphenylactetate-2,3-dioxygenase (HPCD) and catechol-2,3-dioxygenase (C2,3O) (A) DHBD from Pseudomonas sp KKS102 with 2,3-dihydroxybiphenyl bound (PDB code 1eim) (B) Brevibacterium fuscum HPCD (PDB code 1q0c) with homoproto-catechuate bound (C,D) Pseudomonas putida C2,3O (PDB code 1mpy) with 3,4-dimethylcatechol (3,4-DMC) or 3,6-DMC, respectively, docked in the active site Schemes in (E) and (F) illustrate the active site of P putida C2,3O with 3,5-DMC docked in the active site in two different ori-entations Arrows indicate groups at distan-ces between 3 A ˚ and 4.2 A˚ Arrows in bold indicate groups at distances less than the sum of the van der Waals’ radii Hydrogen bonds are shown as dotted lines The schemes of the side chains are shown only when the side chain makes the closest con-tact between the residue and the substrate Lines with round ends indicate stacking between the ring of the substrate and His240 in DHBD (A), His248 in HPCD (B), and His246 in C2,3O (C–F).
Trang 5Based on the above observations, residue Thr249
was substituted in silico with valine, serine, alanine
and glycine, and the molecular contacts of docked
3,6-DMC and 3,5-DMC were reinspected Mutation
T249V does not allow for a reduction of the steric
hin-drance with dimethylated substrates Indeed, the valine
rotamer, which better fits the C2,3O active site, has
one of the methyl groups at approximately the same
position as the Thr249 methyl group that approaches
the substrate On the contrary, the progressive
reduc-tion of the side chain of residue 249 caused by
muta-tion of threonine to serine, alanine and glycine creates
a new cavity (subsite 3¢) adjacent to CH atoms at
posi-tion 6, resulting in the reducposi-tion of steric hindrance
between a methyl group at this position and the
pro-tein The reduction of steric hindrance caused by
mutations was estimated by measuring the radius of
the largest sphere that can be fitted to the active site,
using as center of the sphere the coordinates of the
carbon atom of the methyl group at position 6 of
3,6-DMC bound as shown in Fig 2D The radius
increa-ses from 0.76 A˚—measured for wild-type C2,3O—to
0.98, 1.25 and 1.91 A˚ for T249S C2,3O, T249A
C2,3O, and T249G C2,3O, respectively As the radius
of a methyl group is 1.9–2 A˚, it can be predicted that the ability of the mutants to bind dimethylcatech-ols in a productive conformation should increase pro-gressively, reaching its maximum in mutant T249G C2,3O
Kinetic parameters and regioselectivity of wild-type and mutated C2,3O
To investigate the influence of the side chain of residue Thr249 of C2,3O on the cleavage of 3,5-DMC and 3,6-DMC, the catalytic properties of mutants were studied Based on the results of docking studies, three mutants were produced by site-directed mutagenesis: T249S C2,3O, T249A C2,3O, and T249G C2,3O All of the mutated proteins were active on catechol (Table 1), and had an iron content similar to that of wild-type C2,3O
The regioselectivity of the wild-type and mutant C2,3Os were determined by incubating them with 3-MC or 3,5-DMC, and analyzing the cleavage prod-uct by NMR after extraction with ethyl acetate For all of the C2,3O variants, no aldehydic hydrogen was detected in the product when 3-MC was used as a sub-strate, indicating that other possible products of ring cleavage distal to the methyl group, if present, were below the detection limit (less than about 0.6–0.5% of the cleavage product) On the other hand, when 3,5-DMC was used as a substrate, the 1H spectrum of the product showed a signal at d 9.44, a value consistent with that of an aldehydic hydrogen for a conjugate aldehyde Moreover, no signal that could be assigned
to hydrogen atoms of the product of ring cleavage proximal to the methyl group at position 3 was ever found at the expected field This indicates that the cleavage of 3,5-DMC is distal (‡ 99.0%) to the methyl group at position 3 (Fig 3)
Thus, the analysis above leads to the conclusion that 6-oxohepta-2,4-dienoic acid and 2-hydroxy-3,5-dimethyl-6-oxohexa-2,4-dienoic acid (Fig 3) are the sole or main products of 3-MC and 3,5-DMC ring cleavage, respectively (the NMR spectra of the clea-vage products are shown in Supplementary Fig 1) The kinetic parameters of wild-type C2,3O were determined on purified 3,5-DMC and 3,6-DMC (Table 1) The Kmvalues were found to be 74 lm and
21 lm, respectively, which are approximately 50 and
14 times higher than that measured on catechol More-over, the kcat values were found to be very low, 0.36 s)1 for 3,5-DMC and 0.66 s)1 for 3,6-DMC These values are about 0.2–0.5% of that measured
on catechol (180 s)1) Therefore, the low reactivity of
Fig 3 Possible extradiol cleavage reactions for (A)
3,6-dimethyl-catechol (3,6-DMC), (B), 3-methyl3,6-dimethyl-catechol (3-MC) and (C)
3,5-dimethylcatechol (3,5-DMC) (C).
Trang 6C2,3O from P stutzeri towards 3,5-DMC and
3,6-DMC seems to depend on both weak binding and slow
catalysis
Figure 4 shows the kinetic parameters determined at
pH 7.5 using catechol, 3-MC, 3,5-DMC and 3,6-DMC
as a function of the radius of the largest sphere that
can be fitted to the active site of wild-type and
mutated C2,3O as described in the previous section
This is a direct measurement of the volume of subsite
3¢ (Fig 5), and hence of the ability of the enzyme to
bind 3,5-DMC and 3,6-DMC in an orientation similar
to those of catechol and 3-MC
The Kmvalues of catechol and 3-MC (Table 1) show
a regular, progressive increase as the volume of subsite
3¢ increases (Fig 4A) In contrast, the Km values on
3,5-DMC and 3,6-DMC decrease with the increase of
the volume of subsite 3¢ Assuming that the kinetics of
C2,3O follow the Michaelis–Menten relationship, these
results indicate that a reduction of the volume of
resi-due 249 increases the affinity of the enzyme for
3,5-DMC and 3,6-3,5-DMC and decreases the affinity for
smaller substrates
Mutations at position 249 result in large and partly
unexpected variations in the kcatvalues (Table 1) For
the smaller substrates, catechol and 3-MC, the T249S
mutation has little or no effect on the catalytic
con-stants, whereas replacement of the threonine residue
with an alanine or a glycine residue causes a significant
reduction of the kcatvalues with respect to those
meas-ured for the wild-type enzyme; approximately four-fold
and 20-fold for catechol and 3-MC, respectively On
the other hand, the behavior of the mutants is very
different in the case of dimethylcatechols The T249S
mutation causes an increase in the kcatvalues on
dime-thylcatechols with respect to the wild-type enzyme In
the case of 3,5-DMC, the kcat value is about eight
times higher than that of the wild-type enzyme On the contrary, mutations T249A and T249G have no signifi-cant effect on the catalytic constants measured for
Fig 4 Catalytic parameters of wild-type and mutant catechol-2,3-dioxygenases (C2,3Os) measured at pH 7.5 are shown as functions
of the radii of subsite 3¢ shown in Fig 5 (radii are: 0.76, 0.98, 1.25 and 1.91 A ˚ for wild-type, T249S, T249A and T249G C2,3O, respect-ively) Filled circles, catechol; open circles, 3-methylcatechol (3-MC); filled triangles, 3,6-dimethylcatechol (3,6-DMC); open trian-gles, 3,5-DMC For clarity in (B), the k cat ⁄ K m values on catechol and 3-MC and the values on 3,5-DMC and 3,6-DMC are reported on dif-ferent scales—on the left and on the right, respectively.
Table 1 Kinetic parameters of wild-type and mutated catechol-2,3-dioxygenase.
Substrate
Residue at position 249
Trang 73,5-DMC and cause a small decrease in the catalytic
constants on 3,6-DMC
Discussion
Bioremediation techniques are based on the use of
microorganisms to remove hazardous substances, such
as aromatic molecules, from polluted areas [5,6] The
expansion of the catabolic potential of these bacteria
would greatly improve the applicability of these
tech-niques, by increasing the number of molecules that can
be metabolized by the microorganisms ECD specificity
and regioselectivity control the range of molecules that
can be degraded through the catabolic pathways of
bacteria capable of using aromatic hydrocarbons as
growth substrates [14,28,29] Knowledge of the
molecular determinants that direct their substrate
specificity is essential to tailor their active site to
transform a wider range of substrates, hence widening
the ability of the microorganism to grow on aromatic compounds
Members of the different subfamilies of ECD cata-lyze the oxidative cleavage of a very wide range of dihydroxylated aromatic substrates, ranging from the simple ring of catechol to multiple substituted catech-ols and polycyclic molecules [17–23] Despite differ-ences in their specificity, the catalytic residues seem to
be very well conserved Six residues of the active site are completely conserved [36,37]: the three ligands to the catalytic metal (His154, His214, Glu265 in P stut-zeriC2,3O), two histidines that have been suggested to act as acid–base catalysts (His199 and His246), and Tyr255, which is responsible for the correct positioning
of the substrate [18,34,38]
The structures of two DHBDs, an Fe2+-dependent HPCD and an Mn2+-dependent HPCD are available
in their reduced, active forms with the substrate bound
to the active site [34,35] In each of these structures, the substrate is bound similarly to both the catalytic metal and the conserved residues in the active site pocket One of the substrate hydroxyl groups is posi-tioned near the conserved tyrosine residue and is closer
to the metal atom than the other hydroxyl group [34,35] Available data suggest that the hydroxyl group facing the conserved tyrosine is in the anionic form [34,35]
To shed light on the specificity of the enzyme for dimethylcatechols, the information above was used to construct models of the complexes between P stutzeri C2,3O, a member of subfamily 1 ECDs, and different substrates
The models of the complexes indicate that the orien-tation of the substrate in the active site pocket of the C2,3O is very similar to that observed in the structure
of the DHBD and HPCD complexes A closer compar-ison of the X-ray structures and of our models of C2,3O with bound catechols reveals that the residues interacting with the first hydroxyl group that is strongly coordinated to the metal atom, and those interacting with the two faces of the substrate ring, are conserved The polypeptide regions that contact the edge of the ring, however, are variable in the different proteins Thus, it is likely that the determinants of sub-strate specificity reside in these regions
The model of C2,3O with catechol bound in the act-ive site pocket reveals the presence of two small sub-sites, 1¢ and 2¢ (Fig 2C), facing positions 3 and 4 of the substrate ring The volume of these cavities is large enough to accommode methyl substituents at positions
3 and 4, thus providing a molecular scaffold to sup-port C2,3O binding and cleavage of 3,4-DMC Subsite 2¢, which is adjacent to position 4, is slightly smaller
Fig 5 Scheme of possible binding of 3-methylcatechol (3-MC),
3,5-dimethylcatechol (3,5-DMC) and 3,6-DMC to
catechol-2,3-dioxygenase (C2,3O) active site (A,B) Binding of 3-MC and 4-MC,
respectively, to the active site of wild-type Pseudomonas stutzeri
C2,3O (C,E) Two possible orientations for the binding of 3-MC to
the active site of T246G C2,3O (D,F) Binding of 3,5-DMC and
3,6-DMC, respectively, to the active site of T246G C2,3O.
Trang 8than subsite 1¢ facing position 3 of the catechol ring
(Fig 2C) This difference could explain why
4-substi-tuted catechols are more inactivating substrates than
3-substituted catechols [17,18] The region of C2,3O
adjacent to positions 5 and 6 of the catechol ring
pro-vides no space for the binding of substituents at these
positions This may suggest a structural basis for the
fact that C2,3O is not able to cleave catechols with
substituents at positions 3,5, or 3,6 (Fig 2D,E,F) In
fact, a strong 20–70-fold decrease in the affinity of
P stuzeri C2,3O for 3,5-DMC and 3,6-DMC with
respect to unsubstituted catechol and to 3-DMC is
found (Table 1)
The region facing positions 5 and 6 of the catechol
ring is mainly formed by a loop containing residues
246–249 in C2,3O (240–243 in DHBD and 248–251 in
HPCD) (Fig 2) Multiple alignments of ECDs show
that this loop is well conserved within each subfamily
(Supplementary Fig 2) The consensus sequence of the
loop is H-G-(L⁄ I ⁄ V ⁄ F)-T in C2,3Os, H-(T ⁄ A ⁄ S ⁄
P)-N-D in DHBDs and H-G-(V⁄ I ⁄ L)-S in HPCDs
Despite the differences in their primary structures, the
three different types of loop tightly contact positions 5
and 6 of the substrate ring in a very similar fashion
(Fig 2) It should be noted that none of the members
of the C2,3O, DHBD or HPCD subfamilies have been
reported to cleave catechols with substituents at both
positions 3 and 5 or 3 and 6 Members of the
DHpCD subfamily, on the other hand, have been
reported to cleave 3,6-substituted catechols This
sub-family has the loop consensus sequence H-P-(P⁄ T)-S
Unfortunately, there is no available structure for any
member of the DHpCD subfamily that could provide
insight into the contacts between the loop residues
and the substrate Thus, the structure of
2,3-dihydrox-y-p-cumate-3,4-dioxygenase from P putida F1, a
member of the DHpCD subfamily, was modeled with
the substrate bound in the active site using the
struc-ture of DHBD from Burkholderia cepacia LB400
(1kmy [38]), as a template We found (data not
shown) that the loop containing residues 235–238 of
DHpCD, with the sequence H-P-P-S, can assume a
conformation that easily accommodates the
carboxy-late group of the aromatic substrate
dihydroxy-p-cu-mate, whereas the isopropyl group of the substrate
can be housed in a cavity corresponding to subsite 1¢
of C2,3O (Fig 2C) Moreover, the model indicates
that the carboxylate group can hydrogen bond to
Ser238 of the loop (data not shown) The model also
suggests that the active sites of other ECDs could be
enlarged to accommodate 3,6-disubstituted catechols
by inducing small changes to the loop 246–249 (C2,3O
numbering)
The modeling studies of the C2,3O complexes indi-cate that the active site of this enzyme can accommo-date one methyl group from 3,5-DMC or 3,6-DMC in subsites 1¢ or 2¢, but not a second, because of the dif-ferent structure of loop 246–249 of C2,3O (subsite 3¢) with respect to that of the homologous loop 235–238
of DHpCD Thus, the steric hindrance between the second methyl group and loop 246–249 could force the dimethylated substrate to bind in an orientation that is not suitable for efficient catalysis This hypothesis is supported by the low affinity and low catalytic effi-ciency of wild-type C2,3O on 3,5-DMC and 3,6-DMC and by the results we have obtained from the study of C2,3O Thr249 mutants
The Kmvalues in Fig 4A indicate, as expected, that the apparent affinity of dimethylcatechols for C23O increases as the steric hindrance at position 249 decrea-ses Moreover, the 3,6-DMC Km values for wild-type and mutant C2,3O are lower than those measured for 3,5-DMC and are in agreement with the models shown
in Fig 5D,F Figure 5F shows that the two methyl groups of 3,6-DMC are housed in subsites 1¢ and 3¢, whereas in the model of Fig 5D, the methyl groups of 3,5-DMC are housed in subsites 2¢ and 3¢ The smaller volume of subsite 2¢ compared to subsite 1¢ may explain the lower affinity of wild-type and mutant C2,3O for 3,5-DMC with respect to 3,6-DMC
Interestingly, the progressive decrease of the dimen-sion of the residue 249 side chain also causes an increase in the Km values for catechol and 3-MC (Fig 4A) In the case of the smaller side chain, in mutant T249G C2,3O, the Kmvalues are 63 and seven times higher, respectively, than those measured for the wild-type enzyme, suggesting that residue Thr249 might make an energetic contribution to substrate binding Thr249 could contribute to substrate binding either through van der Waals’ contacts as described in Results, or a through a hydrogen bond network, dis-cussed later in this section
Thr249 mutants also give information on factors that control the regioselectivity of C2,3O 3-MC might
be cleaved at two different bonds (Fig 3), yielding two different extradiol cleavage products All known ECDs belonging to subfamilies 1, 2 and 3 catalyze only the proximal cleavage [17,19–23] (Fig 3) It has been reported that this regioselectivity could depend either
on the reactivity of the substrate or on the asymmetry
of the active site that forces the binding of the sub-strate in the monoanionic form [38–41] The decrease
in Km values of mutant T249G C2,3O on 3,5-DMC and 3,6-DMC could indicate that the T249G mutation
is successful in opening a new subsite (subsite 3¢) for methyl binding Thus, the presence of a new cavity in
Trang 9the active site pocket of mutant T249G C2,3O should
allow for the binding of 3-MC in two different
orienta-tions, i.e with the methyl group housed in subsite 1¢
or in subsite 3¢ (Fig 5C,E) As reported in Results, the
formation of the distal cleavage product (Fig 3) has
never been observed, either with T249G C2,3O or with
the other two mutants These data suggest that the
regioselectivity of the cleavage of 3-MC is proximal,
independently of the orientation of the substrate in the
binding site Thus, the regioselectivity of cleavage
would be mainly controlled by the reactivity of the
substrate This could explain the finding that wild-type
C2,3O and its mutants cleave 3,5-DMC only at the
bond proximal to the methyl group at position 5 This
regioselectivity could indicate that the methyl group at
this position is more activating than the methyl group
at position 3 This latter hypothesis is reinforced by
the observation that the kcatvalue of P stutzeri C2,3O
for 4-MC is two times higher than the kcat value for
3-MC [18] Figure 5B,D show that 4-MC and
3,5-DMC could bind in the active sites of wild-type and
T249G C2,3O, respectively, with a similar orientation
Thus, the methyl group at position 4 of 4-MC is
geo-metrically and chemically equivalent to the methyl
group at position 5 of 3,5-MC Consequently, it could
be the reactivity of a substrate that bears a methyl
sub-stituent at an equivalent position—i.e position 4 in
4-MC and position 5 in 3,5-DMC—that controls the
regioselectivity of the extradiol cleavage we have
observed in the case of 3,5-DMC
Finally, the data reported in Table 1 show that
resi-due 249 also strongly influences the kcatvalues
More-over, the variations observed in the kcat values are
significantly larger than those in the Km values; as a
consequence, the kcat and kcat⁄ Kmvalues show similar
trends as a function of steric hindrance at the 3¢
sub-site (Table 1 and Fig 4B)
Mutation T249S increases the kcat and kcat⁄ Km
val-ues on 3,5-DMC and 3,6-DMC, with respect to those
measured using the wild-type enzyme (Table 1 and
Fig 4B) This effect could depend on the relief of the
steric hindrance in the binding of dimethylcatechols at
the active site, which, in turn, might favor a more
suit-able orientation of the substrate for catalysis
How-ever, mutations T249A and T249G cause instead small
variations in kcat and kcat⁄ Km values (Table 1) despite
the fact that their KM values on 3,5-DMC and
3,6-DMC would suggest improved binding with respect to
the wild-type enzyme Moreover, mutation T249S has
little or no effect on the kcat values on catechol and
3-MC (Table 1), whereas mutations T249A and T249G
reduce by four times the kcatvalues on catechol and 20
times those measured on 3-MC (Table 1)
These latter data are quite intriguing, and they sug-gest that the hydroxyl group of Thr249 could play an unsuspected role in catalysis Its direct involvement in the catalytic mechanism is unlikely, given the distance (4 A˚ or greater) between the oxygen atom of the Thr249 side chain and the groups of the substrate directly involved in the reaction An analysis of the active sites of DHBD structures in the presence and in the absence of substrates and of the structure of
P putida C2,3O suggests a possible hypothesis In the DHBD–substrate complex, a water molecule is bound between the carboxylate group of Asp243 and the hydroxyl group of the substrate (Supplementary Fig 3) [34] A solvent molecule is also present in the active site of each protomer of the P putida C2,3O structure [33], bound to the hydroxyl group of Thr249
at a position equivalent to that of the water molecule bound to DHBD residue Asp243 Our modeling stud-ies show that binding of the substrate to the active site
of P putida C2,3O does not displace the water mole-cule, which can bridge the hydroxyl group of residue Thr249 and one of the hydroxyl groups of the sub-strate, as in the DHBD–substrate complex (Supple-mentary Fig 3) Moreover, in the model of C2,3O with 3,6-DMC and 3,5-DMC bound to the active site, the water molecule contacts the methyl group located
in the 3¢ site (about 3 A˚ between the oxygen atom and the carbon atom of the methyl group) As a conse-quence, the possible removal of the water molecule due to mutations T249A and T249G should not make
a significant contribution to the decrease in steric hin-drance between the substrate and the active site This observation and the reduced catalytic efficiency of T249A C2,3O and T249G C2,3O with respect to the wild-type enzyme and to T249S C2,3O would strongly suggest that the bridging solvent molecule plays an important role in catalysis
Experimental procedures
Materials and general procedures
All chemicals were of the highest grade available and were from Amersham Pharmacia Biotech (Amersham, UK), Promega (Madison, WI, USA), New England Biolabs (Bev-erly, MA, USA), Sigma (St Louis, MO, USA), or Appli-Chem GmbH (Darmstadt, Germany)
SDS⁄ PAGE was carried out according to the method of Laemmli [43] Protein concentration was determined colori-metrically with the Bradford reagent [44], using bovine serum albumin as a standard Total iron content and Fe(II) content were determined colorimetrically by complexation with Ferene S [45]
Trang 10Bacterial strains and plasmids
Escherichia colistrain BL21(DE3) and plasmid pET22b(+)
were purchased from Novagen (Madison, WI, USA)
Plas-mid DNA purifications were performed by using the
Qiagen purification kit (Quiagen, Valencia, CA, USA)
Bac-terial transformation was carried out according to the
method of Sambrook et al [46]
The construction of recombinant plasmid pET22b(+)
DXN⁄ C2,3O used for the expression of wild-type P stutzeri
C2,3O and the preparation of C2,3O mutants is described
elsewhere [18]
Construction of the expression vectors coding
for mutant C2,3Os
Mutant C2,3Os were produced by the Kunkel method [47],
sequences of the mutagenic oligonucleotides for T249S,
T249A and T249G were 5¢-GTCTTGCCGTGACTGAG
GCCGTGG-3¢, 5¢-TCTTGCCGTGAGCGAGGCCGTGG
C-3¢ and 5¢-GGTCTTGCCGTGGCCGAGGCCGTGG-3¢,
respectively The clones harboring the desired mutations
pET22b(+)DXN⁄ (T249S)-C2,3O, pET22b(+)DXN ⁄
(T249A)-C2,3O, and pET22b(+)DXN⁄ (T249G)-C2,3O The DNA
sequences of the three clones were verified by sequencing
Expression and purification of C2,3Os
Wild-type and mutant C2,3O were expressed in E coli
strain BL21(DE3), transformed with the appropriate
expression vector, purified and analyzed for quality as
des-cribed previously [18] C2,3Os were stored at) 80 C under
a nitrogen atmosphere
Synthesis and characterization of 3,5-DMC and
3,6-DMC
Synthesis of 3,5-DMC and 3,6-DMC was achieved by a
modification of the procedure described by Pezzella et al
[48]
o-Iodoxybenzoic acid (IBX) was freshly prepared from
2-iodobenzoic acid as already described [49] Solid IBX (2.5
equivalents) was added to a solution of 2,4-dimethylphenol
or 2,5-dimethylphenol (200 mg) in CHCl3⁄ MeOH 3 : 2 v ⁄ v
(40 mL) at ) 25 C A yellow–orange color developed and
the mixture was stirred for 24 h Methanolic NaBH4
(15 mg in 1 mL) was then added at ) 25 C with vigorous
stirring until the color disappeared (usually within 5 min)
Excess NaBH4 was removed by mild acidification with
acetic acid (200–500 lL) The mixture was then washed five
times with equal volumes of a saturated NaCl solution
con-taining 10% sodium dithionite buffered at pH 7.0 with
sodium phosphate Evaporation of the organic layer even-tually yielded 3,5-DMC or 3,6-DMC, which could be separ-ated by preparative TLC (benzene⁄ ethyl acetate ⁄ acetic acid
1 : 1 : 0.01) on silica
1
H (13C) NMR spectra of products were recorded at 400.1 (100.6) MHz using a Bruker DRX) 400 MHz instru-ment fitted with a 5 mm 1H⁄ broadband gradient probe with inverse geometry Impurities were below 1H-NMR detection limits
Spectral data of 3,5-DMC
Pale brown powder UV(MeOH): kmax281 nm ESI(–)⁄ MS
m⁄ z: calculated for C8H9O2 [M–H+] 137.061, determined 137.060 1H-NMR (CDCl3), d (p.p.m.) of selected signals: 2.19 (s, 3H, CH3), 2.21 (s, 3H, CH3), 6.51 (s, 2H), 6.53 (s, 2H)
Spectral data of 3,6-DMC
Pale brown powder UV(MeOH): kmax280 nm ESI(–)⁄ MS
m⁄ z: calculated for C8H9O2 [M–H+] 137.061, determined 137.060 1H-NMR (CDCl3), d (p.p.m.) of selected signals: 2.22 (s, 6H, CH3), 6.61 (s, 2H) On the basis of 1H-NMR and ESI⁄ MS data, it was possible to confirm the structures
of DMC and 3,6-DMC Indeed, in the case of 3,5-DMC, the presence of two aromatic signals at slightly dif-ferent shifts, given the shielding effect of the OH group at position 1, which is positioned para and ortho to hydrogen
3 and hydrogen 5, respectively, is consistent with the struc-ture of catechol In the case of 3,6-DMC, the 1H-NMR spectrum features only one aromatic and one methyl group signal, as expected based on the symmetry of the molecule Also in this case, observed shifts are in agreement with those predicted on the basis of the structure
Determination of regioselectivity on 3-MC and 3,5-DMC
3-MC or 3,5-DMC were added to a solution containing 0.1 mgÆmL)1 of wild-type or mutated C2,3O in 50 mm Tris⁄ HCl, pH 7.0, at 200 lm final concentration After
5 min at 25C, the reaction was stopped by acidification to
pH 4.0 with H3PO4 at 4C, saturated with NaCl, and extracted with ethyl acetate (3· 100 mL) Evaporation of the organic layer eventually furnished a pale yellow oil that was directly characterized by 1H-NMR (solvent CDCl3) About 95–100% of 3-MC and 70–80% of 3,5-DMC were converted, yielding 60–80 lmol of products The determin-ation of the structures of the cleavage products was done only on the basis of the hydrogen atoms bound to sp2 carbon atoms (see Supplementary Fig 1 for details) The signals of hydrogen atoms of methyl groups were not con-sidered, as they do not allow us to discriminate distal and