(Received for publication, September 25, 1995; and in revised form, December 18, 1995)
From the
The N-carbamyl-D-amino-acid amidohydrolase
from Agrobacterium radiobacter NRRL B11291, the enzyme used
for the industrial production of D-amino acids, was cloned,
sequenced, and expressed in Escherichia coli. The protein, a
dimer constituted by two identical subunits of 34,000 Da with five
cysteines each, was susceptible to aggregation under oxidizing
conditions and highly sensitive to hydrogen peroxide. To investigate
the role of the cysteines in enzyme stability and activity, mutant
proteins were constructed by site-directed mutagenesis in which the
five residues were substituted by either Ala or Ser. Only the mutant
carrying the Cys substitution was catalytically inactive,
and the other mutants maintained the same specific activity as the wild
type enzyme. The crucial role of Cys
in enzymatic
activity was also confirmed by chemical derivatization of the protein
with iodoacetate. Furthermore, chemical derivatizations using both
acrylamide and Ellman's reagent revealed that (i) none of the
five cysteines is engaged in disulfide bridges, (ii) Cys
is easily accessible to the solvent, (iii) Cys
and
Cys
appear to be buried in the protein core, and (iv)
Cys
and Cys
seem to be located within or in
proximity of external loops and are derivatized under mild denaturing
conditions. These data are discussed in light of the possible
mechanisms of enzyme inactivation and catalytic reaction.
Optically active D-amino acids have attained a wide
variety of commercial applications as intermediates for the production
of fine chemicals, including -lactam antibiotics, peptide
hormones, and pesticides(1, 2) . In particular, D-phenylglycine and D-p-hydroxyphenylglycine are
among the most important chiral building blocks for the production of
semisynthetic penicillins and cephalosporins such as ampicillin and
amoxicillin.
Several optically active D-amino acids are currently produced in a two-step reaction process starting from D,L-5 monosubstituted hydantoins that are inexpensively synthesized from the corresponding aldehydes(3) . In the first step, the substrate is hydrolyzed by a D-specific hydantoinase to give a D-carbamyl derivative. Subsequently, the carbamyl derivative is converted to the corresponding D-amino acid either by chemical methods (4) or by a second enzymatic step catalyzed by an N-carbamyl-D-amino-acid amidohydrolase (hereinafter carbamylase)(5) . Because chemical methods have high reaction temperatures, low yields, long reaction times, and generate large amounts of waste, the enzymatic hydrolysis of the N-carbamyl derivatives is highly preferred. Indeed, the use of the D-hydantoinase plus carbamylase two-enzyme system is considered one of the most successful industrial applications of enzyme technology.
Several microorganisms expressing both enzymatic activities have been isolated, and the optimal reaction conditions and the biochemical properties of the two enzymes have been studied in some detail(6, 7) . From what has been published so far, it appears that both the activity and stability of the carbamylase are negatively affected by oxidizing conditions, suggesting that one or more cysteine residues are present in the enzyme. Indeed, the sensitivity of the enzyme to oxidizing conditions is one of the most serious drawbacks of the enzymatic D-amino acid production process, and a strict anaerobic regime is required to allow the completion of the substrate to product conversion(8) .
To shed light on the role of the cysteines in the activity and stability of this important industrial enzyme, we decided to study in detail the N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, a strain that is used industrially for D-amino acid production. In this paper we describe the characterization of the recombinant A. radiobacter N-carbamyl-D-amino-acid amidohydrolase expressed in Escherichia coli. In particular, the role of the five cysteines in the activity of the enzyme has been thoroughly investigated using both chemical and genetic methods. In addition, a topological mapping has been undertaken using a chemical approach in which the availability of the cysteine residues to derivatizing agents has been assessed in native and denaturated enzyme.
For all the mutagenesis reactions, the wild type gene was isolated from plasmid pSM637 as an EcoRI-HindIII fragment and cloned in M13mp8. The mutated EcoRI-HindIII fragment was used to replace the wild type gene of pSM637, and the presence of the mutations on the derived plasmids was confirmed by sequence analysis. The mutated plasmids were used to transform E. coli 71/18, and the positive transformants were grown on LB medium supplemented with 20 µg/ml chloramphenicol for the production of the mutant proteins.
Figure 1: Nucleotide sequence and deduced amino acid sequence of N-carbamyl-D-amino-acid amidohydrolase gene from A. radiobacter NRRL B11291. The CNBr protein fragments isolated and sequenced are indicated.
The gene coding for the N-carbamyl-D-amino-acid amidohydrolase was inserted into plasmid pSM214 to give plasmid pSM637 (Fig. 2), which was used for its heterologous expression in E. coli. The enzyme was expressed under the control of a constitutive promoter at a level that on the basis of SDS-PAGE analysis was estimated to be more than 5% of the total soluble proteins (data not shown).
Figure 2: Restriction map of plasmid pSM637 used for the expression of the N-carbamyl-D-amino-acid amidohydrolase. The carbamylase gene is co-transcribed with the chloramphenicol acetyl transferase gene from pC194 using a B.subtilis-E. coli constitutive promoter (9) .
On SDS-PAGE the molecular mass of the
enzyme was estimated to be about 32,000 Da, in good agreement with the
theoretical calculation from the sequence analysis. However, on
Superose 12 HR 10/30 the protein eluted with an apparent molecular mass
of about 67,000 Da, suggesting that the active form of the enzyme is a
homodimer. The recombinant enzyme co-eluted with bovine serum albumine
(67,000 Da) even in the presence of 10 mM -mercaptoethanol, indicating that the two subunits of the
homodimeric structure are not covalently associated (data not shown).
Under prolonged storage at 4 °C in phosphate buffers, the enzyme
progressively lost activity that could be recovered only partially by
treatment with reducing agents such as -mercaptoethanol (data not
shown). The loss of activity paralleled the appearance of insoluble
inactive aggregates in the enzyme preparation, suggesting that covalent
intermolecular reactions took place under the storage conditions used.
Nevertheless the purified enzyme was more stable in the absence rather
than in the presence of
-mercaptoethanol. This can be explained by
the fact that in the presence of air thiol groups have a propensity to
generate hydrogen peroxide and superoxide anions, which in turn can
lead to irreversible enzyme damage(18, 19) .
To
test the sensitivity of the enzyme to hydrogen peroxide, the activity
was measured after incubation either in the presence or in the absence
of HO
. As shown in Fig. 3A, the
enzyme was fully inactivated after 15 min of incubation with 0.1 mM H
O
. The enzyme was protected from
H
O
inactivation when a sufficient amount of
catalase was included in the reaction mixture before
H
O
addition (Fig. 3B). The
sensitivity of the enzyme to H
O
suggested that
at least one cysteine residue takes part in the catalytic reaction.
Figure 3:
Effect of HO
on N-carbamyl-D-amino-acid amidohydrolase in the
presence and absence of catalase. A, the enzyme was incubated
with different concentrations of hydrogen peroxide for 15 min
(0-0.2 mM) at room temperature, and the residual
activity, expressed as percentage of initial activity, was determined
using the standard assay conditions. B, the enzyme was
incubated at room temperature for 15 min in the presence of both
H
O
(0.1 mM) and catalase (0-5
units), and the residual activity was
determined.
To experimentally test whether one of the five cysteines present in
the carbamylase from A. radiobacter NRRL B11291 is involved in
catalysis, we expressed in E. coli five mutants of the enzyme
in which each of the cysteine residues was replaced with alanine.
Substitution of Cys by alanine resulted in a completely
inactive enzyme under the standard assay conditions, whereas
substitution of any of the other cysteines had no effect on enzyme
activity. Substitution of Cys
with serine also resulted
in complete inactivation of the enzyme. A number of double mutants were
also generated (Cys
/Cys
, Cys
/Cys
, Cys
/Cys
,
Cys
/Cys
, and
Cys
/Cys
). Taken together, these data
clearly indicated that only Cys
is strictly required for
enzymatic activity.
Figure 4:
Reverse phase HPLC separation of the CNBr
fragments obtained after N-carbamyl-D-amino-acid
amidohydrolase derivatization with acrylamide in denaturing conditions.
The CNBr reaction was performed in 70% trifluoroacetic acid. The arrows indicate the Cys-containing fragments. In particular,
the 13.5-min peak corresponds to fragment 170-184, which contains
Cys, the peak that elutes at 14.3 min corresponds to
fragment 185-220 containing Cys
, and the 25.8-min
peak is the 240-304 fragment containing Cys
,
Cys
, and Cys
5.
Treatment of the enzyme with 0.2 M iodoacetate before the denaturation and the alkylation step
resulted in modification of available cysteine thiols, and the Edman
degradation product of the modified cysteines (PTH S-carboxymethylcysteine) can be distinguished from PTH
Cys-Pam. Only Cys
was identified as PTH S-carboxymethylcysteine (Table 2), whereas all other
cysteines were recovered as PTH Cys
-Pam, indicating that only
Cys
is exposed to the aqueous solvent. Because the
iodoacetate-treated enzyme was fully inactive (data not shown), we
concluded that in agreement with the site-directed mutagenesis
experiments, Cys
must be involved in the catalytic
reaction.
In an attempt to further investigate
the spatial organization of the cysteine residues, native N-carbamyl-D-amino-acid amidohydrolase was treated
with 1 M acrylamide, fully denatured with 5 M guanidinium hydrochloride, and finally derivatized with the
thiol-reacting Ellman's reagent. When the chemical modification
of the five cysteines was analyzed by peptide sequencing, we found that
cysteines 172, 243, and 279 had been alkylated by acrylamide
(identified as PTH Cys-Pam), whereas Cys
and
Cys
were identified as the dithiothreitol adducts of
dehydroalanine (Table 2), indicating that they had been modified
by Ellman's reagent after full denaturation of the enzyme.
Considering that a 1 M solution of acrylamide is a weak
protein denaturant, the experiments described above can be interpreted
as indicating that Cys and Cys
are exposed
to the solvent by partial unfolding of the protein and are thus located
within or in proximity of external loops. On the other hand,
Cys
and Cys
are expected to be buried in
the protein core and become accessible to the thiol-reacting compounds
only after full denaturation.
The carbamyl-D-amino-acid amidohydrolases are
important enzymes for the industrial production of D-amino
acids, some of which are key intermediates for the synthesis of the
-lactam antibiotics. The relevant role of these enzymes in the
pharmaceutical industry prompted us to study in more detail the
biochemical properties of the carbamyl-D-amino-acid
amidohydrolase from A.radiobacter, a bacterial strain that,
due to its ability to synthesize large quantities of this enzyme
together with hydantoinase, is currently used for the production of p-OH-phenylglycine and phenylglycine starting from the
corresponding hydantoines(5, 20) . In particular, we
focused our attention on the role of cysteine residues on the activity
of the enzyme.
Sequence analysis of the carbamylase gene revealed that it encodes a protein of 32,000 Da containing five cysteines. On the basis of gel filtration and SDS-PAGE analyses, the enzyme was found to be organized in a nondisulfide bonded homodimeric structure.
The organization in more than one subunit appears to be a typical feature of amidohydrolases. For example, the N-carbamyl-D-amino-acid amidohydrolase from Comamonas sp. E222c, which has been recently characterized and whose N-terminal sequence is highly homologous to the corresponding region of the A. radiobacter enzyme, was proposed to have a trimeric structure with three identical subunits(7) . Furthermore, the bacterial amidohydrolases that catalyze the hydrolysis of asparagine and glutamine are active as tetramers of identical protein chains with molecular weights in the range of 34,000-36,000 per monomer(21, 22) . Finally, the mature form of the penicillin acylase from E. coli is a periplasmic 80,000-Da heterodimer having the A and B chains of 209 and 566 amino acids, respectively(23) .
Using chemical methods
we proved that the five cysteines of the enzyme are not engaged in
intramolecular disulfide bonds. Sequence analysis of the CNBr-generated
fragments containing the cysteine residues after treatment of the
denatured enzyme with thiol-reacting acrylamide revealed that all the
five cysteines were alkylated by the acrylamide. These data indicating
the absence of disulfides in the carbamylase are consistent with the
cytoplasmic nature of the enzyme. As a consequence of the presence of
free thiols groups, the purified enzyme progressively lost activity
that could be partially recovered when reducing agents such as
dithiothreitol or -mercaptoethanol were added to the enzyme
preparation. However, somewhat surprisingly, we found that the shelf
life of the enzyme was prolonged when the reducing agents were not
included in the storage buffers. To reconcile these two apparently
contradicting results, we hypothesized that the main
enzyme-inactivating agent is hydrogen peroxide. It is known that in the
presence of air the thiol groups have a propensity to generate
H
O
and superoxide anions, which in turn can
lead to irreversible enzyme damage(18, 19) .
Our
experiments demonstrating the high sensitivity of the N-carbamyl-D-amino-acid amidohydrolase to
HO
supports this hypothesis. We suggest that
the pathway for enzyme inactivation proceeds through various degrees of
thiol oxidation, from inter- or intramolecular disulfide bonds to more
oxidized products such as cysteic acid(24) . The reducing
agents exert two opposite effects. On one side they are beneficial,
protecting the enzyme against reversible inactivation probably caused
by disulfide bond formation. On the other hand, however, they are
detrimental to enzyme activity because of their propensity to generate
highly toxic H
O
. A direct consequence of this
hypothesized mechanism for enzyme inactivation is that at least one of
the cysteine residues of the enzyme takes part in the catalytic
reaction.
The experiments described in this work clearly
demonstrated that this is indeed the case. The Cys
Ala substitution produced a fully inactive enzyme, at least as judged
by our standard assay, which can detect enzymes with specific activity
10,000-fold lower than the wild type. Similar conclusions were obtained
following a chemical approach. Cys
was the only cysteine
that under nondenaturing conditions could be chemically modified by
either iodoacetate or Ellman's reagent. After modification, the
enzyme was totally inactive.
The involvement of a cysteine in the
enzymatic activity fits with the type of chemical reaction catalyzed by
carbamylase. Among the amide bond-hydrolyzing enzymes, many utilize
either the hydroxyl group of serine and threonine side chains or the
thiol group of cysteine as a nucleophile to attack the scissile bond.
Typical examples are the serine and cysteine
proteases(25, 26) , the amydohydrolases, which
catalyze the hydrolysis of asparagine and glutamine to their acidic
forms(22, 27, 28) , and the E. coli penicillin acylase responsible for the conversion of penicillin G
to 6-aminopenicillanic acid(23) . Interestingly, the N-carbamyl-D-amino-acid amidohydrolase from A.
radiobacter shares about 25% identity with the aliphatic amidases
of both Pseudomonas aeruginosa and Rhodococcus
erythropolis(29, 30) with the highest degree of
homology being found in the region surrounding and including
Cys.
Grouping the A. radiobacter carbamylase
in the ``cysteine and serine proteases family'' implies that
(i) the reaction proceeds through the formation of an acyl-enzyme
intermediate and (ii) a neighboring residue such as histidine or lysine
should serve as a base to enhance the nucleophilicity of
Cys. Both properties can be experimentally tested. In
particular, we recently developed a rapid screening procedure able to
identify on agar plates carbamylase-deficient mutants. We are utilizing
such screening protocol to shed light on the amino acid residues
crucial for the enzymatic activity.
It has been shown that in some cysteine-dependent enzymes the active site cysteine can be substituted for serine without completely destroying the enzyme activity. For example, the substituted thymidylate synthases of both E. coli and bacteriophage T4 retain 0.02 and 0.07% activity of the wild type enzymes, respectively (31, 32) .
Our
Cys
Ser mutant was at least 4 orders of magnitute
less active than the wild type, indicating that the carbamylase has
more structural and catalytic constrains than other cysteine-dependent
enzymes. The resolution of the three-dimensional structure of the
enzyme will be of great help to shed light on the details of the
catalytic reaction. In this context, the availability of large
quantities of pure enzyme will facilitate future protein
crystallization experiments.
An interesting aspect of the work
presented here is that by using different protocols for the
derivatization of the thiol groups, including treatment of the enzyme
under native, mild denaturing, and strong denaturing conditions, it has
been possible to have some hints as to the topological position of the
five cysteines present in the molecule. The data clearly show that the
active site Cys is readily accessible to the solvent,
Cys
and Cys
are buried in the protein core,
and Cys
and Cys
are probably located near
external loops. These last two cysteines may be the ones involved in
the formation of intermolecular disulfide bridges that we found to
occur under oxidizing conditions. If this is the case, the replacement
of Cys
and Cys
with other amino acids
should be beneficial for the enzyme stability.