Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
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544 KAUL ET AL.
and reaction mechanism of nitrile metabolising enzymes will lead to the improved
properties such as greater enzyme activity, increased tolerance to harsh compounds
and improved thermostability of the biocatalysts used in commercial processes.
The development of novel nitrile degrading enzymes with increasing regio-,
stereo-, and chemo-specificities is also required for bioremediation. With continuing
development in techniques related to screening, cultivation, protein and genetic
engineering it is possible to isolate novel organisms of extremophilic character or
improve the functions of known organisms. Though recent advances have broadened
the scope of the potential applications of these versatile biocatalysts, further
application-oriented studies are required to fully exploit their biotechnological
potential.
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CHAPTER 31
ASPARTASES: MOLECULAR STRUCTURE,
BIOCHEMICAL FUNCTION AND BIOTECHNOLOGICAL
APPLICATIONS
TOMOHIRO MIZOBATA AND YASUSHI KAWATA
∗
Department of Biotechnology, Faculty of Engineering, and Department of Biomedical Science,
Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori
University, Koyama-Minami, Tottori, Japan
∗
kawata@lemon.bio.tottori-u.ac.jp
1. INTRODUCTION
The experimental finding that a unique enzyme in bacteria mediates an equilibrium
between L-aspartic acid, fumaric acid and ammonia was first reported by Quastel
and Woolf (Quastel and Woolf, 1926) using cultures of E. coli shifted to resting
phase by the addition of propyl alcohol or toluene. Further characterization deter-
mined that this enzyme was capable of catalysing the deamination of L-aspartate,
and it was named aspartase in 1929 (Cook and Woolf, 1928). Its isolation was
successfully achieved from cell extracts of Pseudomonas fluorescens (Virtanen and
Tarnanen, 1932), and was subsequently followed by a series of studies performed
by Ellfolk describing the initial characterization of this activity which involved its
partial purification (Ellfolk, 1953a) and the determination of its substrate specificity
(Ellfolk, 1954) and chemical modification (Ellfolk, 1953b).
The purification of aspartase from E. coli cells was first reported by Rudolph
and Fromm (Rudolph and Fromm, 1971) and involved multiple purification steps.
Subsequent refinements in the protocol gradually succeeded in improving both
the yield and purity of the enzyme, and an important addition to the purification
protocol, dye-ligand chromatography using Red-A agarose and elution with 1 mM
L-aspartate (Karsten et al., 1985), allowed researchers to obtain pure aspartase in
very high yields. The cloning of the structural gene of this enzyme was successful for
a variety of bacterial species including E. coli (Guest et al., 1984) and Pseudomonas
549
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 549–565.
© 2007 Springer.
550 MIZOBATA AND KAWATA
1 11 21 31 41 51
E. coli M------SNN IRIEEDLLGT REVPADAYYG VHTLRAIENF YISNNKISDI PEFVRGMVMV
P. fluorescens MISVMSSAAS FRTEKDLLGV LEVPAQAYYG IQTLRAVNNF RLSGVPISHY PKLVVGLAMV
Cytophaga KUC-1 M-------GS TRKEHDFLGE LDIPNHLYYG IQTFRAVENF NITGIPISKE PLFIKALGYV
B. subtilis M---LNGQKE YRVEKDFLGE KQIEADVYYG IQTLRASENF PITGYKI--H EEMINALAIV
Bacillus YM55-1 M------NTD VRIEKDFLGE KEIPKDAYYG VQTIRATENF PITGYRI--H PELIKSLGIV
* . * *.*:** :: . *** ::*:** :** ::. * :: .: *
61 71 81 91 101 111
E. coli KKAAAMANKE LQTIPKSVAN AIIAACDEVL NNGKCMDQFP VDVYQGGAGT SVNMNTNEVL
P. fluorescens KQAAADANRE LGQLSERKHA AISEACARLI -RGDFHEEFV VDMIQGGAGT STNMNANEVI
Cytophaga KUC-1 KKAAALANKD CGRLDPKIAE AICYGSDQVI -AGKFDQEFV SDLIQGGAGT SVNMNANEVI
B. subtilis KKAAALANMD VKRLYEGIGQ AIVQAADEIL -EGKWHDQFI VDPIQGGAGT SMNMNANEVI
Bacillus YM55-1 KKSAALANME VGLLDKEVGQ YIVKAADEVI -EGKWNDQFI VDPIQGGAGT SINMNANEVI
*::** ** : : * .. .:: *. ::* * ****** * ***:***:
121 131 141 151 161 171
E. coli ANIGLELMGH QKGEYQYLNP NDHVNKCQST NDAYPTGFRI AVYSSLIKLV DAINQLREGF
P. fluorescens ANIALEAMGH QKGEYQYLHP NNDVNMAQST NDAYPTAIRL GLLLGHDALL ASLDSLIQAF
Cytophaga KUC-1 ANIGLEYLGH KKGDYNFLHP NNHVNCSQST NDAYPSAFRI ALYLKMESFI KTLEGLEVAF
B. subtilis GNRALEIMGH KKGDYIHLSP NTHVNMSQSQ NDVFPTAIHI STLKLLEKLL KTMEDMHSVF
Bacillus YM55-1 ANRALELMGE EKGNYSKISP NSHVNMSQST NDAFPTATHI AVLSLLNQLI ETTKYMQQEF
.* .** :*. :**:* : * * .** .** **.:*:. :: . :: : . : *
181 191 201 211 221 231
E. coli ERKAVEFQDI LKMGRTQLQD AVPMTLGQEF RAFSILLKEE VKNIQR-TAE LLLEVNLGAT
P. fluorescens AAKGAEFSHV LKMGRTQLQD AVPMTLGQEF RAFATTLGED LARLKTLAPE LLTEVNLGGT
Cytophaga KUC-1 VANGEEFKSV LKMGRTQLQD AVPMTLGQEF RSYATTIGED VRRLKE-AQS LVLEINMGAT
B. subtilis KQKAQEFHSV IKMGRTHLQD AVPIRLGQEF EAYSRVLERD IKRIKQ-SRQ HLYEVNMGAT
Bacillus YM55-1 MKKADEFAGV IKMGRTHLQD AVPILLGQEF EAYARVIARD IERIAN-TRN NLYDINMGAT
:. ** : :*****:*** ***: ***** .::: : .: : .: : . : ::*:*.*
241 251 261 271 281 291
E. coli AIGTGLNTPK EYSPLAVKKL AEVTGFPCVP AEDLIEATSD CGAYVMVHGA LKRLAVKMSK
P. fluorescens AIGTGINADP RYQALAVQRL ATISGQPLVP AADLIEATSD MGAFVLFSGM LKRTAVKLSK
Cytophaga KUC-1 AIGTRVNAPE GYPEICVNYL AKEVGIPLTL SPDLIEATVD TGAYVQIMGT LKRTAVKISK
B. subtilis AVGTGLNADP EYIKQVVKHL ADISGLPLVG ADHLVDATQN TDAYTEVSAS LKVCMMNMSK
Bacillus YM55-1 AVGTGLNADP EYISIVTEHL AKFSGHPLRS AQHLVDATQN TDCYTEVSSA LKVCMINMSK
*:** :*: * .: * * * * : .*::** : ..:. . . ** :::**
301 311 321 331 341 351
E. coli ICNDLRLLSS GPRAGLNEIN LPELQAGSSI MPAKVNPVVP EVVNQVCFKV IGNDTTVTMA
P. fluorescens ICNDLRLLSS GPRTGINEIN LPARQPGSSI MPGKVNPVIP EAVNQVAFQV IGNDLALTMA
Cytophaga KUC-1 ICNDLRLLSS GPRTGFNEIN LPARQPGSSI MPGKVNPVIP EVVNQTCFYV IGQDLTVTMA
B. subtilis IANDLRLMAS GPRAGLAEIS LPARQPGSSI MPGKVNPVMA ELINQIAFQV IGNDNTICLA
Bacillus YM55-1 IANDLRLMAS GPRAGLSEIV LPARQPGSSI MPGKVNPVMP EVMNQVAFQV FGNDLTITSA
*.*****::* ***:*: ** ** *.**** **.*****:. * :** .* * :*:* :: *
361 371 381 391 401 411
E. coli AEAGQLQLNV MEPVIGQAMF ESVHILTNAC YNLLEKCING ITANKEVCEG YVYNSIGIVT
P. fluorescens AEGGQLQLNV MEPLIAFKIF DSIRLLQRAM DMLREHCIVG ITANEARCRE LVEHSIGLVT
Cytophaga KUC-1 AEAGQLQLNV MEPVIAFAMF TSLDYLSNAI QTLIDKCIIG ITANVDHCYN MVMNSIGIVT
gi|142518|gb|AA SEAGQLELNV MEPVLVFNLL QSISIMNNGF RSFTDNCLKG IEANEKRMKQ YVEKSAGVIT
Bacillus YM55-1 SEAGQFELNV MEPVLFFNLI QSISIMTNVF KSFTENCLKG IKANEERMKE YVEKSIGIIT
:*.**::*** ***:: :: *: : . : ::*: * * ** * :* *::*
421 431 441 451 461 471
E. coli YLNPFIGHHN GDIVGKICAE TGKSVREV-V LERGLLTEAE LDDIFSVQNL MHPAYKAKRY
P. fluorescens ALNPYIGYEN ATRIARIALE SGRGVLEL-V REEGLLDDAM LDDILRPENM IAPRLVPLKA
Cytophaga KUC-1 QLNPILGYEI SASIAGEALK MNKSVHEIVV VERKLITQEK WDEIYSLDNL INPKFITK--
B. subtilis AVNPHLGYEA AARIAREAIM TGQSVRDL-C LQHDVLTEEE LDIILNPYEM TKPGIAGKEL
Bacillus YM55-1 AINPHVGYET AAKLAREAYL TGESIREL-C IKYGVLTEEQ LNEILNPYEM THPGIAGRK-
:** :*:. . :. . ...: :: : :: : : * :: *
481
E. coli TDESEQ
P. fluorescens ------
Cytophaga KUC-1 ------
B. subtilis LEK---
Bacillus YM55-1 ------
ASPARTASES 551
fluorescens (Takagi et al., 1986), and overproduction of the gene product in suitable
hosts followed soon after. At present, researchers are able to obtain highly purified
samples of aspartase from a variety of organisms using the combination of overpro-
ducing strains and dye-ligand chromatography.
A comparatively recent development has been the isolation and characteri-
zation of aspartases from a number of extremophiles, for example the moderately
thermophilic Bacillus species Bacillus sp. YM55-1 (Kawata et al., 1999) and
the psychrophile Cytophaga sp. (Kazuoka et al., 2003). The aspartases isolated
from these exotic sources possess numerous unique characteristics compared to
the aspartases from mesophilic sources. Details will be given in the following
sections.
2. BASIC CHARACTERISTICS OF ASPARTASES
Aspartases from different organisms share a number of common structural charac-
teristics. The enzymes from Pseudomonas fluorescens (Takagi et al., 1986), E. coli
(Takagi et al., 1985), Bacillus sp. YM55-1 (Kawata et al., 1999) and Cytophaga
(Kazuoka et al., 2003) are all homotetrameric enzymes with subunit molecular
weights of around 50∼52 kDa. Amino acid sequence homologies are extensive
(Fig. 1), and 45% ∼ 50% identity occurs between sequences from various sources
(Sun and Setlow, 1991; Kawata et al., 2000). The Bacillus subtilis and Bacillus
sp. YM55-1 aspartases exhibit 71% identity (Kawata et al., 2000). In addition, the
aspartases are members of a larger family of enzymes which includes the type
II fumarases (Acuna et al., 1991) and the adenylosuccinate lyases (Woods et al.,
1988). High levels of amino acid sequence homology are also shared between these
enzymes.
As determined from gene sequencing the E. coli aspartase subunit is composed
of 478 amino acid residues and has a molecular weight of approximately 52 kDa
(Takagi et al., 1985). The theoretical pI of this enzyme is 5.2, and a T
m
of 55
C
was determined in thermal unfolding experiments monitored by intrinsic tyrosine
fluorescence (Kawata et al., 1999). The enzyme retains 50% of its initial activity
after a 4 minute incubation at this temperature but is completely inactivated after
30 min (Kawata et al., 1999).
Figure 1. Alignment of the amino acid sequences of aspartases. The numbering scheme shown is
arbitrary and does not correspond to the residue numbers of any specific type of aspartase. Alignment
of five known amino acid sequences are shown: E. coli, Escherichia coli (Burland et al., 1995);
P. fluorescens, Pseudomonas fluorescens (Takagi et al., 1986); Cytophaga KUC-1, Cytophaga sp.
KUC-1 (Kazuoka et al., 2003); B. subtilis, Bacillus subtilis (Sun and Setlow, 1991); and Bacillus
YM55-1, Bacillus sp. YM55-1 (Kawata et al., 2000). In the case of Escherichia coli aspartase the
first 15 residues of the derived amino acid sequence were omitted to reflect the actual amino acid
sequence of the aspartase protein. Alignment analysis was performed using an online version of the
MAFFT sequence alignment program (Katoh et al., 2005)
552 MIZOBATA AND KAWATA
2.1. Enzymatic Characteristics
The enzymatic characteristics of the E. coli aspartase are very notable in many
respects. Firstly, the specificity of this enzyme for its substrates, L-aspartate
and fumarate, is extremely high; to date, only the suicide substrate L-aspartate
-semialdehyde has been discovered that is efficiently deaminated by this
enzyme (Yumoto et al., 1982; Schindler and Viola, 1994). Aspartase is inhibited
quite efficiently however, by a variety of compounds that mimic the struc-
tural properties of L-aspartate, such as D- and L-malate, succinate, and N -acetyl
L-aspartate. Falzone and co-workers have proposed that the presence of a
carboxylate or equivalent functional group in the 1 and 4 positions of the backbone
carbon chain of the substrate is important for binding to the aspartase binding site
(Falzone et al., 1988).
Another interesting characteristic of this enzyme is its behaviour at various pH
and the effects of different divalent cations. In the absence of a divalent cation the
pH dependence of aspartase displays a bell-shaped curve with decreased activity at
the acidic and alkaline extremes and a single activity maximum at pH ∼6 (Karsten
and Viola, 1991). However, in the presence of 10 mM Mg
2+
, the activity at alkaline
pH is greatly increased and a second activity maximum is observed at pH ∼8. The
presence of divalent cations acts as an activator of aspartase activity at high pH.
Further studies revealed the nature of this cation-derived activation to be a relatively
complicated phenomenon. In this pH region, the substrate saturation curves display
a distinctly sigmoidal character, indicative of a cooperative mechanism involving
multiple subunits of the aspartase tetramer (Ida and Tokushige, 1985; Karsten
et al., 1986). When this characteristic was examined it was found that L-aspartate
acts in cooperation with divalent cations to allosterically enhance the activity of
aspartase (in both directions of the reversible deamination-amination reaction). Thus
L-aspartate is not only a substrate of aspartase, but in cooperation with divalent
cations it is also a potent activator. Similar behaviour has been confirmed for the
related protein fumarase C, for which very high concentrations of the substrate
malate result in enhanced activity (Beeckmans and Van Driessche, 1998; Rose and
Weaver, 2004). pH dependent enzyme function, divalent cation action, and the
presence of a secondary substrate binding site which enhances the activity of both
the forward and backward reactions, all combine to confer aspartase an exquisitely
complex catalytic mechanism.
The enzymatic mechanism of aspartase was elucidated by studies which involved
the characterization of a specific inhibitor of aspartase and the analysis of isotope
effects using specifically labelled substrate molecules. In 1980 Porter and Bright
identified 2-nitro-3-aminopropionate to be a very potent inhibitor of the aspartate
deamination activity of aspartase (Porter and Bright, 1980). This compound was
found to inhibit aspartase most strongly under pH conditions where the aci-acid
form was most stable. This aci-acid form closely resembles a carbanion interme-
diate state of L-aspartate with a hydrogen atom removed, and this was taken as
strong evidence for the formation of a carbanion intermediate during the reaction
cycle. Detailed studies were performed on the kinetic mechanism of the aspartase
ASPARTASES 553
from Hafina alvei using deuterium-substituted and
15
N-substituted substrates (Nuiry
et al., 1984). This study determined that the kinetic mechanism is initiated by the
binding of a divalent cation followed by binding of the activator aspartate molecule.
Binding of cation and aspartate occur separately, rather than through a pre-formed
cation-aspartate complex. A significant isotope effect attributable to
15
N substitution
was also observed which suggested that the rate limiting step of the deamination
reaction was the scission of the C-N bond of aspartate. The effects of this inhibitor,
taken together with additional isotope effects observed with specifically deuterated
forms of L-aspartate, indicate the following enzyme mechanism for aspartase (Fig. 2
Yoon et al., 1995).
The deamination of L-aspartate is initiated by the removal of a proton from
C3 of L-aspartate. The subsequent intermediate state is stabilized in the carbanion
form, as suggested by the effects of 2-nitro-3-aminoprionate. The next, rate limiting,
step of the overall reaction is the removal of the ammonium group NH
3
. The
detached NH
3
molecule is then protonated by a general acid group located in the
vicinity to form NH
+
4
, which is the actual leaving group. The release of the products
fumarate, NH
+
4
and the divalent cation are thought to occur in a random manner.
This acid/base mechanism of catalysis requires three properties associated with
the enzyme structure for successful catalysis to occur (Fig. 2): a general base that
mediates the initial removal of the proton from C3; a residue that stabilizes the
carbanion intermediate state, most likely through charge stabilization; and a general
Figure 2. Proposed catalytic mechanism of aspartase (Yoon et al., 1995). The mechanism is depicted
clockwise from the upper left-hand corner
554 MIZOBATA AND KAWATA
acid group that assists the removal of the ammonia group. These three factors are
considered to be assigned to specific residues located in the vicinity of the aspartase
active site. The identification of these residues proved to be exceedingly difficult,
however. Initial studies that utilized chemical modification to target specific amino
acid types as well as detailed analysis of the pH dependence of aspartase catalysis
suggested that cysteine (Mizuta and Tokushige, 1975; Ida and Tokushige, 1985),
lysine (Karsten and Viola, 1991) and histidine (Ida and Tokushige, 1984) residues
were important participants in the catalytic mechanism. Armed with this infor-
mation, a number of groups performed site-directed mutagenesis studies on these
three residues, in some cases focusing on those that were highly conserved among
various species, and in the case of cysteine (Chen et al., 1996) an exhaustive substi-
tution analysis of all of the relevant residues in the amino acid sequence. The results
from these studies indicated that none of the cysteine residues were essential for
catalysis. Similar studies also ruled out a conserved histidine residue as an active
participant in catalysis (Viola, 2000). Finally, two highly conserved lysine residues,
Lys 327 and Lys 55, were found (through replacement by arginine residues) to be
very important for enzyme function (Saribas et al., 1994). Subsequently, the loss
of activity in the Lys55Arg mutant was found to be due to aggregation and inacti-
vation of the aspartase that could be partially reversed by incubation in denaturant
(Jayasekera and Viola, 1999). However, Lys 327 was determined to be important
in the initial binding of substrate and also in communications between the active
site and the pH-dependent activation site.
2.2. X-ray Crystal Structure of Aspartase
In 1997 a high resolution crystal structure of E. coli aspartase, shown in Fig. 3,
was reported (Shi et al., 1997). This was the second structure of a member of
the aspartase-fumarase superfamily to be elucidated, the first being that of E. coli
fumarase (Weaver et al., 1995). The aspartase subunit consists of three distinct
sequentially arranged domains, denoted 1, 2 and 3 (Fig. 3a). The central domain,
domain 2, consists almost entirely of -helical elements and forms the interface
for subunit association. Domains 1 and 3 display a more dynamic nature in the
crystallography analyses and are thought to form the actual active centres of the
enzyme. The final arrangement of the aspartase tetramer may be described as
consisting of a central subunit interface region and four active centres arranged
regularly on both sides of this central interface (Fig. 3b).
Three of the four subunits in aspartase are thought to contribute important amino
acid residues to form a given active site (Shi et al., 1997). However, the actual
position of the active site was not readily apparent in the initial structural deter-
mination of the apo form of aspartase. Data from the mutagenesis studies outlined
above that suggested that Lys 327 was important for effective binding of substrate
(Saribas et al., 1994) localized the active site in the vicinity of this lysine residue.
A number of amino acid residues found within a 15 Å radius of Lys 327 were listed
as candidates for catalytic participation (Jayasekera et al., 1997). Of five residues
identified in this study, one, Arg 29, has been implicated in substrate binding, and