Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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ASPARTASES 555

Figure 3. X-ray crystal structure of Escherichia coli aspartase (Shi et al., 1997). (a) Domain composition

of the E. coli aspartase subunit. (b) Structure of the aspartase tetramer

another, Ser 143, has been implicated to act as a general acid catalyst that mediates

the protonation of the departing ammonium group. Still missing from this molecular

view of the aspartase active site is the residue that acts as a general base to remove

a proton from C3 of L-aspartate to form the carbanion intermediate. Although a

number of candidates have been probed to date, notably His 26 and Asp 10, neither

of these residues satisfied the criteria of a general base catalyst.

3. BACILLUS sp. YM55-1 ASPARTASE

Relatively recently a new member of the aspartase family was isolated from

an aerobic, moderately thermophilic bacterium, Bacillus sp. YM55-1 (Kawata

et al., 1999). This aspartase displayed a number of characteristics that were

different from those of the mesophilic aspartases. Firstly, the specific activity of

Bacillus sp. YM55-1 aspartase was significantly (∼5 fold) higher than that of

the other mesophilic aspartases under identical conditions (assayed at 30



C). This

enhancement was much more pronounced when the enzyme was assayed at its

optimum temperature of 55



C, yielding a specific activity of 2200 units/mg protein,

which is among the highest specific activities known for an aspartase. The stability

of this aspartase against thermal and chemical denaturation was also significantly

better than that of E. coli aspartase. This has been attributed to increased numbers

of intersubunit hydrogen bonds and ion pairs in the Bacillus sp. YM55-1 enzyme

(Fujii et al., 2003). Another interesting difference between the Bacillus sp. YM55-

1 aspartase and other mesophilic aspartases is that the former does not display

enhanced activity at alkaline pH in the presence of divalent cations, nor does it

undergo substrate activation by L-aspartate. The enzyme kinetics of Bacillus sp.

YM55-1 aspartase are hyperbolic at all pH values tested, and cooperativity is notably

lacking.

Cloning and overproduction of Bacillus sp. YM55-1 aspartase in E. coli (Kawata

et al., 2000) and crystallization/determination of its structure (Fujii et al., 2003)

556 MIZOBATA AND KAWATA

followed soon after (Fig. 4). The general structure of Bacillus sp. TM55-1 aspartase

is essentially identical to that of E. coli aspartase and E. coli fumarase: a central

subunit interface consisting mainly of -helices, and active sites flanking this

central structure (compare Figs. 3 and 4). Upon closer examination a number of

additional similarities, and some notable differences, are apparent between these

three molecular structures.

A docking simulation of aspartate bound to Bacillus sp. YM55-1 aspartase was

performed in order to ascertain the orientation and possible active site residues in this

enzyme (Figs.5 and 6; Fujii etal., 2003). Lys 327, theresidue that binds the -carboxyl

group in E. coli aspartase, corresponds to Lys 324 in the Bacillus sp. structure, and this

residue was postulated to act in a similar role in this thermostable enzyme. The amino

group was arranged to be bound through three separate residues (Fig. 6): Asn142 (E.

coli Asn 145), His 188 (E. coli Gln 191) and either Gly 98 (E. coli Gly 100) or Thr 101

(E. coli Thr 104). Docking was based on the finding that residues homologous to these

threeinE. coli fumarase (Ser98, Thr 100, Asn141 and His 188)were found complexed

with a water molecule. This water molecule is regarded as being very important in the

catalytic mechanism of fumarase, and an analogy may be derived that implicates these

residues in aspartase catalysis as well. The -carboxyl group of docked L-aspartate

was found to interact with Ser 140 (E. coli Ser 143) in the completed docking model

(Fig. 6). This residue is located at the N-terminal end of a helix dipole (helix 6 in the

subunit structure) and the positive charge provided by this dipole is thought to assist

in binding.

Figure 4. X-ray crystal structure of Bacillus sp. YM55-1 aspartase (Fujii et al., 2003). (a) The domain

composition of the aspartase subunit, oriented in a similar manner to that of the E. coli aspartase subunit

shown in Fig. 3a. (b) Structure of the Bacillus sp. YM55-1 aspartase tetramer. In this figure the tetramer

was arranged in a manner such that the relative positions of the domains in a given subunit is apparent.

Images were created using a coordinate file of the Bacillus aspartase tetramer (provided generously by

Drs. T. Fujii and Y. Hata, Kyoto University)

ASPARTASES 557

Figure 5. Close-up view of the active site of Bacillus sp. YM55-1 aspartase with an L-aspartate molecule

in the postulated binding site. The left image shows a view of the aspartase tetramer with different

shading assigned to individual subunits. Following the naming convention of Fujii et al. (2003) individual

subunits are shaded dark to light in the following sequence: subunit a, subunit b, subunit c. Subunit d,

which does not participate in the formation of the active site, is the most lightly shaded. The right image

shows a zoomed view of the portion of the left panel. The relative orientation of the two images is

identical. The amino acids that are postulated to participate in the catalytic mechanism of aspartase are

drawn in the zoomed view in either stick or ball-and-stick form, and the relative shading of the amino

acid residues denotes the subunit from which each residue is derived. The docked L-aspartate residue is

shown in thick stick form

From numerous studies of members of the aspartase-fumarase superfamily it has

been suggested that residues at the position corresponding to His 188 in Bacillus

sp. YM55-1 aspartase – modeled to interact with the amino group of L-aspartate in

Figure 6. Stereo view of the active site of Bacillus sp. YM55-1 aspartase, reprinted from Fujii et al. (2003)

558 MIZOBATA AND KAWATA

Figs. 5 and 6 – not only participate in substrate binding but also actively participate

in the catalytic mechanism as a general acid catalyst (Blanchard and Cleland, 1980;

Weaver and Banaszak, 1996; Weaver et al., 1997; Wu et al., 1998; Lee et al.,

1999). His 188 is highly conserved among most of the members of the fumarase

superfamily, including the aspartase from Bacillus sp. YM55-1. However, that is

not the case in E. coli aspartase where a glutamine residue occurs at this position

(Gln 191 in E. coli). Although this difference in a crucial residue may indicate

that this position in the active site of the aspartase-fumarase superfamily is not an

essential functional position, the homology between Bacillus sp. YM55-1 aspartase

and the other members of the fumarase family may warrant a more detailed scrutiny

of this interesting residue.

Another residue that was proposed to be an active participant in the catalytic

mechanism of the E. coli aspartase was Ser 143 (Ser 140 in Bacillus sp. YM55-1

aspartase). Ser 143 was postulated to be the general acid residue that protonates the

ammonia leaving group (Jayasekera et al., 1997). The position of Ser 140 in Bacillus

sp. YM55-1 aspartase is, however, comparatively distant from the candidate active

site region and the role of general acid catalyst seemed difficult to achieve in this

enzyme (Fujii et al., 2003). Instead, Ser 140 is postulated to bind the -carboxyl

group of L-aspartate, thus filling an important role in substrate binding for this

residue (Fig. 6). An alternate residue that could assume the role of general acid

catalyst in place of Ser 140 was not readily apparent in the Bacillus sp. YM55-1

structure. The detailed understanding of the catalytic mechanism of Bacillus sp.

aspartase is therefore far from complete.

With regard to the substrate activation effect that is seen in both E. coli

aspartase and fumarase but not in Bacillus sp. YM55-1 aspartase, structural

comparison has revealed that a site corresponding closely to the B-site in

E. coli fumarase (where a L-malate molecule was found to be bound by crystal

structure analysis (Weaver and Banaszak, 1996)) also exists in the Bacillus sp.

YM55-1 and E. coli aspartases (Fig. 7). A detailed comparison of this region

(corresponding to residues 123–128 in Bacillus sp. aspartase) showed that the

structure of Bacillus sp. YM55-1 aspartase more closely resembles the structure

of E. coli fumarase C (Fig. 7a) rather than the apo structure of E. coli aspartase

(Fig. 7b). It may be that the Bacillus sp. aspartase is locked in an active confor-

mation similar to that seen for ligand-bound E. coli fumarase. An additional

striking finding was that in Bacillus sp. YM55-1 aspartase, a tyrosine side chain

(Tyr 126) was found in a position which partially occluded this activation site

(Fig. 7a). This may be the reason why the enzyme does not display a prominent

substrate activation effect and is locked in a permanently active conformation.

Related to this finding, Fujii and co-workers showed that the structure of apo

E. coli aspartase differs markedly from the structures of Bacillus sp. YM55-1

aspartase and E. coli fumarase (liganded form) in the regions corresponding to

residues 40–45, 80–84, 228–241 and 265–272. A detailed examination of these

regions may yield additional clues about substrate-activated catalysis in E. coli

aspartase.

ASPARTASES 559

Figure 7. Superimposed comparisons of the active site and activator binding site regions of E. coli

aspartase, E. coli fumarase and Bacillus sp. YM55-1 aspartase reprinted from Fujii et al. (2003). In both

panels the docked L-aspartate molecule is shown by a thick ball-and-stick model. (a) Superposition of

Bacillus sp. YM55-1 aspartase (wire model) with E. coli fumarase (thin ball-and-stick model). Note the

extension of Tyr126 into the activation site region. (b) Superposition of E. coli aspartase (wire model)

with E. coli fumarase (thin ball-and-stick model)

4. MOLECULAR ENGINEERING OF ASPARTASE

AND INDUSTRIAL APPLICATIONS

In industry, aspartase is used mainly in the enzyme-mediated production of

L-aspartate, an important starting compound for generating food additives and

560 MIZOBATA AND KAWATA

artificial sweeteners. A survey of patents awarded for the application of aspartase

reveals a number of interesting methods that have been used to alter the activity of

the enzyme with a view toward its utilization in various industrial processes. In this

section, we summarize the numerous methods that have been used in the industrial

application of aspartase, and also provide some hints toward future efforts.

4.1. C-terminal Deletion of Amino Acid Residues

Early studies on aspartase discovered that the specific activity of this enzyme

could be enhanced by brief treatment with certain proteases such as trypsin or

subtilisin (Yumoto et al., 1980; Yumoto et al., 1982). Analysis showed that

C-terminal truncation was responsible for this enhancement of activity. Jayasekera

and co-workers (Jayasekera et al., 1997) undertook a detailed analysis of C-terminal

deletion mutants of E. coli aspartase and found that a mutant aspartase truncated

after Tyr 472 displayed a greater than two-fold enhancement of k

cat

and a minimal

increase in the K

, yielding an enzyme with enhanced activities. The results of this

study have been patented for use in industrial applications (US Patent 5,993,807).

A similar patent (US Patent 6,015,704) was later awarded to Tsai and co-workers

who combined the effects of C-terminal truncation with the substitution of selected

histidine residues by glutamine (His 25, 123, 421 or 463). These two patents open

the way toward the creation of novel aspartase proteins with improved enzymatic

activities suitable for industrial applications.

4.2. Random Mutagenesis and Directed Evolution

The difficulties in identifying the specific residues that are important for aspartase

function have led to the utilization of random mutagenesis as a viable method for

obtaining aspartases having enhanced activities. Zhang and co-workers success-

fully used this method to obtain a mutant E. coli aspartase whose specific activity

was increased 4-fold and which also showed slightly improved thermostability

(Zhang et al., 1993). The characteristics of these enzymes were compared to those

of another mutant aspartase produced by site-directed mutagenesis, Lys126Arg.

Although both enzymes displayed similar specific activities, it is not known

whether the enzyme derived from random mutagenesis is similar or identical to

the Lys126Arg mutant. Another enhanced aspartase enzyme was obtained using

a directed evolution approach comprising random mutagenesis followed by gene

shuffling (Wang et al., 2000). The enzyme obtained had the following amino acid

substitutions; Asn217Lys, Thr233Arg and Val367Gly. Alteration of these three

amino acids resulted in an enzyme with a k

cat

that was increased 28-fold

compared to wild type, a K

that was decreased 4.6 fold, and improved thermosta-

bility such that 61% of its initial activity was retained after a 30 min incubation

at 50



C. Under similar conditions the wild type enzyme only retained 17% of its

activity. This improved aspartase would be an ideal candidate for application in

industry.

ASPARTASES 561

4.3. Isolation of Thermostable Aspartases from Thermophilies

and Psychrophiles

As outlined above, the aspartase isolated from Bacillus sp. YM55-1 possesses many

characteristics that are desirable for industrial applications, including high specific

activity and thermostability. Other examples of thermostable aspartases have also

been reported. Kimura and co-workers have been awarded a patent (US Patent

4,391,910) outlining their discovery of thermophilic aspartases isolated from various

Bacillus species such as Bacillus aminogenes and Bacillus thermoaminophilus. The

enzymes described in the patent are characterized by very high specific activity,

much like that of the aspartase from Bacillus sp. YM55-1. However, unlike the latter,

the aspartases described in the patent are activated by the presence of divalent cations

such as magnesium ion. This interesting difference in enzymatic characteristics may

suggest that the finer characteristics of aspartases may differ considerably even

among members of the same genus.

Recently, the isolation of a novel thermostable aspartase from a marine

psychrophile, Cytophaga sp. KUC-1 (Kazuoka et al., 2003) has been reported

whose optimal temperature of growth is 15



C. This aspartase displays enzymatic

characteristics that are similar to those of the E. coli enzyme, such as activation by

and L-aspartate. However, the thermostability of the Cytophaga enzyme is

considerably higher: about 80% of its activity is retained after a 60 min incubation at



C. Thus the enzyme from Cytophaga KUC-1 represents an interesting example

of an aspartase whose characteristics are a composite of those derived from aspar-

tases from mesophilic and thermophilic sources. A comparative study of these three

enzyme types should be very enlightening as regards the elucidation of the various

molecular mechanisms which underlie the functional aspects of aspartase.

4.4. Immobilization of Aspartase and Aspartase-producing Bacteria

within Solid Gel Matrices

Industrial production of L-aspartate using aspartase and aspartase-producing

organisms is an excellent example of the successful application of an enzymatic

activity in industrial processes. Since the early 1970’s various methods have been

developed to utilize the activity of aspartase to produce L-aspartate in bulk quantities

(Sato and Tosa, 1993). Early attempts involved the immobilization of purified

aspartase enzyme in polyacrylamide lattices (Tosa et al., 1973) and was followed by

immobilization of aspartase-producing bacteria themselves in gel matrices such as

polyurethane (Fusee et al., 1981) and polyacrylamide. The immobilization of living

cells in gel matrices provides certain advantages over immobilizing pure enzymes

such as enhanced stability of the enzymatic activity and the saving of labour

costs associated with enzyme purification. Future applications in this direction may

involve utilizing bacteria that produce enhanced forms of the aspartase, for example

E. coli cells expressing the aspartase from Bacillus sp. YM55-1.

562 MIZOBATA AND KAWATA

4.5. Other Studies

A number of interesting studies on aspartase provide hints toward future work on

applications for this enzyme. Early studies by Murase and co-workers have shown

that aspartase is partially active in a dimeric state induced by the addition of a

denaturant (Murase et al., 1993). In support of this finding, recent work by Kong

and co-workers succeeded in constructing a novel form of aspartase in which two

original subunits of E. coli aspartase were joined by a suitable peptide linker to

produce a dimer-mimicking, monomeric form of the enzyme that was active (Kong

et al., 2002). This monomeric form of aspartase displayed improved stability at

high temperatures compared to the wild type enzyme. Such approaches toward

stabilizing the structure and ultimately the activity of aspartase represent a novel

alternate method for improving the high efficiency of this enzyme.

Another interesting approach that has been tested involves the construction of

hybrid and/or chimeric proteins. By ingenious utilization of gene fusion and in vivo

selection for the desired activity Sheng et al. succeeded in constructing a 74-kDa

polypeptide hybrid enzyme combining the activities of -aspartyl dipeptidase (85%

of wild type) and aspartase (87% of wild type) into a single polypeptide (Sheng

et al., 2005). As an added bonus, this novel hybrid enzyme displayed improved

thermostabilities with regard to both activities. This astonishing example of protein

engineering is indicative of the potential that still remains to be tapped in aspartase.

We too have recently begun to study various chimeric constructs of aspartase genes

with a view to generating novel aspartases having improved thermostabilities and

enzymic activities.

5. CONCLUDING REMARKS

The accumulation of knowledge on the structure and function of aspartase provides

us with a detailed view of the mechanisms that underlie the unique functional

aspects of this intriguing enzyme. Nevertheless, experimentation on aspartase never

fails to produce exciting new developments that promise to lead to previously

unsuspected applications and novel concepts. Exploration of the functional and

structural aspects of aspartase is work in progress, and future studies will further

deepen our understanding of this enzyme.

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