Tài liệu Báo cáo khoa học: Isolation and molecular characterization of a novel D-hydantoinase from Jannaschia sp. CCS1 docx

Isolation and molecular characterization of a novel
D-hydantoinase from Jannaschia sp. CCS1
Yuanheng Cai
1
, Peter Trodler
2
, Shimin Jiang
1
, Weiwen Zhang
3
, Yan Wu
1
, Yinhua Lu
1
, Sheng Yang
1
and Weihong Jiang
1,4
1 Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai, China
2 Institute of Technical Biochemistry, University of Stuttgart, Germany
3 Center for Ecogenomics, Biodesign Institute, Arizona State University, Tempe, AZ, USA
4 Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
Optically pure d-orl-amino acids are used as inter-
mediates in several industries. d-amino acids are
involved in the synthesis of antibiotics, pesticides,
sweeteners and other biologically active peptides.
l-amino acids are used as feed and food additives, as
intermediates for pharmaceuticals, cosmetics and pesti-
cides, and as c hiral c ompounds in organic synthesis [1–4].
Among them, d-p-hydroxyphenylglycine (d-p-HPG)

attracts the most attention as it can be used as the side
chain for production of semi-synthetic b-lactam antibi-
otics, such as amoxicillins and cephalosporins [2].
There are currently two main approaches used to
Keywords
hydantoinase; Jannaschia sp. CCS1;
saturated mutagenesis; structural analysis;
substrate binding pocket
Correspondence
W. Jiang, Key Laboratory of Synthetic
Biology, Institute of Plant Physiology and
Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences,
Shanghai, 200032, China
Fax: +86 21 54924015
Tel: +86 21 54924172
E-mail:
(Received 4 February 2009, revised 15 April
2009, accepted 27 April 2009)
doi:10.1111/j.1742-4658.2009.07077.x
Hydantoinases (HYDs) are important enzymes for industrial production of
optically pure amino acids, which are widely used as precursors for various
semi-synthetic antibiotics. By a process coupling genomic data mining with
activity screening, a new hydantoinase, tentatively designated HYD
Js
, was
identified from Jannaschia sp. CCS1 and overexpressed in Escherichia coli.
The specific activity of HYD
Js
on d,l-p-hydroxyphenylhydantoin as the

substrate was three times higher than that of the hydantoinase originating
from Burkholderia pickettii (HYD
Bp
) that is currently used in industry. The
enzyme obtained was a homotetramer with a molecular mass of 253 kDa.
The pH and temperature optima for HYD
Js
were 7.6 and 50 °C respec-
tively, similar to those of HYD
Bp
. Kinetic analysis showed that HYD
Js
has
a higher k
cat
value on d,l-p-hydroxyphenylhydantoin than HYD
Bp
does.
Homology modeling and substrate docking analyses of HYD
Js
and HYD
Bp
were performed, and the results revealed an enlarged substrate binding
pocket in HYD
Js
, which may allow better access of substrates to the cata-
lytic centre and could account for the increased specific activity of HYD
Js
.
Three amino acid residues critical for HYD

Js
activity, Phe63, Leu92 and
Phe150 were also identified by substrate docking and site-directed muta-
genesis. Application of this high-specific activity HYD
Js
could improve the
industrial production of optically pure amino acids, such as d-p-hydroxy-
phenylglycine. Moreover, the structural analysis also provides new insights
on enzyme–substrate interaction, which shed light on engineering of hydan-
toinases for high catalytic activity.
Abbreviations
DCase, N-carbamoyl-
D-amino acid amidohydrolase; DHU, dihydrouracil; D-p-HPG, D-p-hydroxyphenylglycine; D,L-p-HPH, D,L-p-
hydroxyphenylhydantoin; HDT, hydantoin; HYD, hydantoinase; HYD
Bp,
hydantoinase from Burkholderia pickettii; HYD
Js,
hydantoinase from
Jannaschia sp. CCS1; PDB, protein data bank; SGLs, stereochemistry gate loops.
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3575
obtain optically pure amino acids, namely chemical
and enzymatic syntheses. Chemical synthesis gives
racemic mixtures of amino acids of low yield and is
not environmentally friendly. In contrast, enzyme-
based biological methods are good alternatives to
obtain various d-orl-amino acids with high optical
purity.
Hydantoinases (HYDs) are commonly used in the
industrial production of optically pure amino acids.
According to the EC nomenclature, d-hydantoinase is

an alternative name for dihydropyrimidinase (EC
3.5.2.2) [3]. In a hydantoinase-based process,
hydantoin or its 5-monosubstituted derivatives are
enantioselectively hydrolyzed into corresponding
N-carbamoyl-d-amino acids, which can be further con-
verted into corresponding d-amino acids by chemical
or enzymatic decarbamoylation [4–6]. Dihydropyrimi-
dinases catalyze the reversible hydrolytic ring opening
of the amide bond in 5- or 6-membered cyclic diamides
[1,4]. They are involved at the second step in the
reductive pathway of pyrimidine degradation in many
organisms [7–10]. Depending on the substrate stereose-
lectivity and specificity, hydantoinases are often classi-
fied as d-, l- or non-selective [11]. Significant research
efforts have focused on the use of hydantoinases to
produce optically pure amino acids [5,12–14].
Hydantoinases are known to be present in certain
microorganisms [8,15]. Three approaches have been
used to identify them in the past. The initial approach
to accessing hydantoinases involved screening and iso-
lating naturally occurring enzymes possessing hydan-
toin-hydrolyzing activity from microbes, and using
them to produce optically pure amino acids [4,16–18].
The second approach involved accessing hydantoinase
genes by cloning, and expressing them heterologously
in more efficient hosts. In a previous study, a d-hydan-
toinase gene was cloned from Burkholderia pickettii
(HYD
Bp
) and heterologously expressed in Escherichi-

a coli [19]. The HYD
Bp
hydantoinase was highly
homologous to the hydatoinase from Agrobacterium
sp. KNK712 that has been used in industry for the
production of d-amino acids [20]. The structure of
HYD
Bp
was also determined, and its catalytic active
site was found to consist of two metal ions and six
highly conserved amino acid residues. Although
HYD
Bp
shares only moderate sequence similarity with
d-HYDs from Thermus sp. [21,22] and Bacillus stearo-
thermophilus [23], whose structures have recently been
solved, their overall structures and the catalytic active
sites are strikingly similar [19]. The third approach was
made possible due to the availability of whole genome
sequences of a large number of microbes, which pro-
vide an increasingly rich source of information to
assist in the isolation of desired new enzymes [1]. This
approach was demonstrated recently by Kim et al.
[24], who identified a putative hydantoinase gene from
the E. coli genome database. After high-level expres-
sion, they were able to demonstrate that the putative
hydantoinase was a d-stereo-specific phenylhydan-
toinase. Previously, no hydantoinase activity had been
found in E. coli, and therefore it is unlikely that an
attempt would have been made to isolate such enzymes

from these bacteria [1,24]. In this study, using coupled
genome database mining with activity screening, we
have successfully identified a new hydantoinase from
the Jannaschia sp. CCS1 genome, designated HYD
Js
.
Biochemical analysis showed that HYD
Js
has a specific
activity approximately three times higher than that of
HYD
Bp
when using d,l-p-hydroxyphenylhydantoin
(d,l-p-HPH) as the substrate. Further characterization
revealed that this higher specific activity was mainly
due to the enlarged substrate pocket in HYD
Js
, which
allows better access of catalytic domains to d,l-p-HPH
and a high overall catalytic rate. The study provides
new insights on enzyme–substrate interaction, suggest-
ing possibilities for further engineering of the HYD for
high catalytic activity. In addition, the high specific
activity HYD
Js
can be readily applied for industrial
production of optically pure amino acids.
Results
Genome database mining and identification of
putative

D-hydantoinase genes
The whole genome sequences of various microorgan-
isms available in various public databases have
provided an additional source for identifying d-hydan-
toinases with high catalytic activity. In this study, an
approach combining genomic database mining and
activity screening was utilized. First, all putative
enzymes that were predicted to have hydantoinase
activity but have not been characterized before were
checked within the BRENDA database. Second, the
selected sequences were subject to catalytic domain
analysis and alignment with HYD
Bp
. The typical
characteristics of hydantoinases were checked for
selected sequences, including cyclic amidohydrolase
super-family, and strictly conserved residues for metal
binding and substrate coordination, such as the four
histidines, one aspartic acid and one carboxylated
lysine that are crucial for hydantoinase activity [19,25]
(Fig. 1). Third, the hydantoinases that have higher
identity (> 70%) with HYD
Bp
were eliminated to
avoid repetitive characterization of enzymes similar to
those previously identified, and the less homologous
A novel high-activity D-hydantoinase from Jannaschia sp. CCS1 Y. Cai et al.
3576
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS
Fig. 1. Multiple sequence alignment of hydantoinases from various organisms. Sequences of known hydantoinases from Burkholderia picket-

tii (HYDbp), B. thermocatenulatus GH2 (HYDbth), Pseudomonas sp. KNK003A (KNK 003A) and Bacillus sp. KNK245, plus 12 other putative
hydantoinases obtained by genomic mining. These are labeled 1–12, and are enzymes from Jannaschia sp. CCS1, Pseudomonas fluorescens
PfO-1, Streptomyces coelicolor A3(2), Burkholderia cenocepacia AU 1054, Chlorobium phaeobacteroides BS1, Desulfitobacterium hafniense
DCB-2, Jannaschia sp. CCS1, Polaromonas sp. JS666, Moorella thermoacetica ATCC 39073, Arthrobacter sp. FB24, Burkholderia sp. 383 and
Rubrobacter xylanophilus DSM 9941, respectively. The secondary structure elements are shown above the sequences based on the structure
of HYD
Bp
. The strictly conserved residues are shaded black, and the residues relevant to metal ion binding are indicated by filled stars.
Y. Cai et al. A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3577
Fig. 1. (Continued).
A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1 Y. Cai et al.
3578
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS
putative hydantoinases were subjected to activity
screening. Of 36 predicted hydantoinases, 12 putative
hydantoinase sequences were selected based on these
criteria, which included hydantoinases from Jannaschia
sp. CCS1 (YP_510647), Pseudomonas fluorescens PfO-1
(Q3KAM5), Streptomyces coelicolor A3(2) (O69809),
Burkholderia cenocepacia AU 1054 (Q1BGK8), Chloro-
bium phaeobacteroides BS1 (Q4AGB4), Desulfitobacte-
rium hafniense Y51 (YP_518039), Jannaschia sp. CCS1
(ABD54405), Polaromonas sp. JS666 (Q12FP8), Moo-
rella thermoacetica ATCC 39073 (Q2RGZ6), Arthrob-
acter sp. FB24 (Q2RGZ6), Burkholderia sp. 383
(Q39PA8) and Rubrobacter xylanophilus DSM 9941
(Q1ASG7). However, only three putative hydantoinase

sequences were cloned and tested for activity in this
study due to lack of genomic DNA for other strains:
these are the hydantoinases from Jannaschia sp.
CCS1, P. fluorescens PfO-1 and S. coelicolor A3(2).
Cloning and expression of the putative hyd
genes
The deduced open reading frames of putative hyd
genes were PCR-amplified from the genomic DNA of
the corresponding organisms. The PCR fragments of
the coding regions of HYDs were inserted in-frame
into pET28a, resulting in HYDs that were His-tagged
at the N-terminus. Expression of HYDs was per-
formed using E. coli BL21(DE3) harboring corre-
sponding HYD expression plasmids. HYD
Bp
was
simultaneously expressed and used as a control. The
whole-cell activity of the cloned HYDs was checked
against d,l-p-HPH. The results showed that only the
HYDs from Jannaschia sp. CCS1 (HYD
Js
) and Pseu-
domonas fluorescens PfO-1 (HYD
Pf
) were able to
hydrolyze d,l-p-HPH. SDS–PAGE analyses of whole-
cell extracts and the supernatant and precipitate frac-
tions are shown in Fig. 2. It was found that E. coli
BL21(DE3) ⁄ pHYD
Js

and E. coli BL21(DE3) ⁄ pHYD
Pf
produced a predominant band with an apparent
molecular mass of approximately 56 kDa, which is
consistent with the calculated mass of the His-tagged
translational product of the corresponding hyd genes.
The monomer size of HYD
Bp
was similar to that of
other hydantoinases, which are mostly between 50 and
60 kDa [4]. It is noteworthy that overexpression of
HYD
Js
resulted in the formation of inclusion bodies in
the precipitate fraction, which may lead to low activity
of whole-cell extract, while HYD
Bp
and HYD
Pf
were
mainly expressed in soluble fraction under the experi-
mental conditions used (Fig. 3).
Purification and specific activities of HYDs
HYD
Js
was purified to homogeneity from E. coli
BL21(DE3) ⁄ pHYD
Js
by one-step affinity column chro-
matography. The purity was estimated to be greater

than 98%, as determined by SDS–PAGE analysis
(Fig. 3). Purification of HYD
Bp
and HYD
Pf
from
E. coli BL21(DE3) ⁄ pHYD
Bp
and E. coli BL21(DE3) ⁄
pHYD
Pf
was also performed. Their specific activities
for hydrolyzing d,l-p-HPH were also determined and
compared. The specific activity of HYD
Js
was about
three times higher than that of HYD
Bp
, and five times
higher than that of HYD
Pf
(Table 1). As the activity
of HYD
Pf
at the whole-cell level was lower than that
of HYD
Bp
, even though it seems to be more soluble
than HYD
Bp

(data not shown), we concluded that the
specific activity of HYD
Pf
may be less than that of
HYD
Bp
, and that it may not be worth further investi-
gation. Therefore, the rest of the study focused on
Fig. 2. SDS–PAGE analysis of HYD expression. ppt, precipitate
fraction; sup, supernatant fraction. The molecular weight standard
(lane M) is indicated on the right.
Fig. 3. Purification of HYD
Bp
and HYD
Js
. tot, total proteins; ppt,
precipitate fraction; sup, supernatant fraction; puri, purified proteins;
M, molecular weight standards. For the molecular weight stan-
dards, the bands from top to bottom correspond to 116.0, 66.2,
45.0, 35.0, 25.0 and 18.4 kDa, respectively.
Y. Cai et al. A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3579
characterization and evaluation of HYD
Js
from
Jannaschia sp. CCS1.
Characterization of HYD
Js
To explore the possible cause of the higher specific

activity for conversion of d,l-p-HPH to N-carbamoyl-
p-hydroxyphenylglycine by HYD
Js
, the kinetic
parameters of HYD
Js
and HYD
Bp
were comparatively
determined (Table 2). The mean K
m
values were simi-
lar for both enzymes, but HYD
Js
had a much higher
k
cat
, suggesting a higher turnover rate of HYD
Js
compared to HYD
Bp
.
The pH and temperature dependence of HYD
Js
activ-
ity were measured (Fig. 4). The results revealed an opti-
mal temperature of HYD
Js
of 50 °C for hydrolyzing
d,l-p-HPH, which is the same as that for HYD

Bp
[19].
The optimal pH for the hydrolytic activity of HYD
Js
was 7.6, slightly lower than that for HYD
Bp
(pH 9.0,
unpublished data). In a two-step process to produce
d-p-HPG, N-carbamoyl-d-amino acid amidohydrolase
(DCase) catalyzes stereo-specific transformation of
N-carbamoyl-p-hydroxyphenylglycine into its corre-
sponding d-p-HPG. As we have previously identified a
DCase for hydrolyzing N-carbamoyl-p-hydroxyphenyl-
glycine with optimal activity at pH 7.0, HYD
Js
has an
advantage over HYD
Bp
for coupling with an immobi-
lized DCase for combined conversion of d,l-p-HPH to
d-p-HPG as the optimal pH of two enzymes are very
close.
To test the substrate specificity, eight other substrates,
namely dihydrouracil (DHU), hydantoin, d,l-p-HPH,
dimethylhydantoin, phenylhydantoin, diphenylhydan-
toin, 5-(hydroxymethyl)uracil, benzylhydantoin and iso-
propylhydantoin, were also tested with HYD
Js
. Activity
measurements showed that DHU was the best substrate

among them (Table 3), and can be hydrolyzed ten times
more efficiently than d,l-p-HPH can.
Previous reports suggested that native HYDs from
divergent sources usually occur as either homodimers
or homotetramers [4]. Gel filtration analysis of native
HYD
Js
indicated a molecular mass of about 253 kDa,
and, as the subunit molecular mass of the His-tagged
recombinant HYD
Js
was estimated to be 56 kDa, these
results suggest that HYD
Js
occurs as a homotetramer
in solution.
Homology structural modeling of HYD
Js
A homology model of the structure of HYD
Js
was
generated to further investigate the structural basis for
the higher activity of HYD
Js
compared to HYD
Bp
.
The Z-score for the homology model HYD
Js
based on

use of the manual template Dictyostelium discoideum
Table 1. Specific activities of HYD
Bp
, HYD
Js
and HYD
Pf
with D,L-p-
HPH as the substrate.
Enzymes
Specific activity
(unitsÆmg
)1
)
HYD
Bp
1.9 ± 0.4
HYD
Js
8.2 ± 0.7
HYD
Pf
1.4 ± 0.2
Table 2. Kinetic parameters for HYD
Js
and HYD
Bp
with D,L-p-HPH
as the substrate. Parameters were calculated by the Eadie–Hofstee
method. Values are the mean ± SD of three independent experi-

ments.
Enzyme K
m
(mM) k
cat
(s
)1
) k
cat
⁄ K
m
(mM
)1
Æs
)1
)
HYD
Js
18.0 ± 1.0 4.6 ± 0.2 0.25
HYD
Bp
14.1 ± 1.3 0.54 ± 0.03 0.038
Fig. 4. Temperature and pH dependence of HYD
Js
. (A) Tempera-
ture ⁄ activity profile of purified HYD
Js
; (B) pH ⁄ activity profile of
purified HYD
Js

.
A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1 Y. Cai et al.
3580
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS
dihydropyrimidinase (PDB accession number 2FTW)
was )8.82 by ProSA [26], which was better than that
for the model generated by automatically choosing dif-
ferent templates, which was a minimum of )8.74. It
was proposed that the active center of a d-hydantoin-
ase is formed by three stereochemistry gate loops
(SGLs), which constitute a hydrophobic binding
pocket [27]. The three SGLs of HYD
Js
, i.e. SGL1,
SGL2 and SGL3, correspond to residues 60–71, 91–99
and 151–161, respectively. On the basis of the homol-
ogy model, the SGLs of HYD
Js
and HYD
Bp
(PDB
accession number 1NFG) were superimposed and com-
pared. The SGL1 and SGL2 of both enzymes are very
similar, with only small differences for backbone
atoms, but there is a greater difference between the
SGL3 of the two enzymes. In HYD
Js
, the size of the
substrate binding pocket and the entrance to the active

site are larger compared to those of HYD
Bp
, making it
more accessible for larger substrates (Fig. 5).
Docking analysis of substrate on the active site
of HYD
Js
The substrate binding pocket in the active site was inves-
tigated based on the homology model to obtain more
information on substrate binding in HYD
Js
. The orien-
tation of the substrates was not resolved experimentally
as no competitive inhibitor is known for hydantoinases,
therefore the productive transition states of hydantoin,
d-p-HPH, l-p-HPH and DHU were docked into the
active site of HYD
Js
[28] to simulate the mode of sub-
strate binding. The results suggest that the substrate
binding pocket accommodates substrates with small side
chains better than those with large ones, which is in
accordance with the specific activity of HYD
Js
against
the tested substrates (Table 3).
Identification of active-site residues of HYD
Js
On the basis of fitting d,l-p-HPH as a target substrate
into the active site of HYD

Js
, the amino acid residues
interacting with the substrate were deduced. Four
possible amino acid residue positions that are critical
in the substrate binding pocket, Phe63, Leu92, Phe150
and Tyr153, were revealed to be related to substrate
binding and recognition of d-p-HPH and l-p-HPH by
HYD
Js
, preferring d-p-HPH as substrate. The bulky
side chains of Phe63, Leu92, Phe150 and Tyr153 were
Table 3. Substrate specificity of HYD
Js
. The relative rate of hydro-
lysis of various substrates is shown as a percentage of the rate at
which HYD
Js
hydrolyzes dihydrouracil. ND, enzyme activity corre-
sponding to less than 1% of the rate at which HYD
Js
hydrolyzes
dihydrouracil.
Substrates
Relative
activity (%)
Dihydrouracil 100
Hydantoin 18.7
D,L-p-hydroxyphenylhydantoin 7.2
Dimethylhydantoin 1.4
Phenylhydantoin 45.0

Diphenylhydantoin ND
5-(hydroxymethyl)uracil ND
Benzylhydantoin ND
Isopropylhydantoin ND
A
B
Fig. 5. Homology model of HYD
Js
. (A) Docking of D-p-HPH to the
HYD
Js
active site. (B) The SGLs of HYD
Bp
are shown as magenta
lines and those of HYD
Js
are shown in green. The transition state
of
D-p-HPH is shown in turquoise, and the four histidines coordinat-
ing the metal ions are shown in white. Phe63, Leu92, Phe150 and
Tyr153, which formed close contacts with the exocyclic substituent
of
D-p-HPH, are shown as green spheres, and the two metal ions
are shown in gray. The red spheres show the oxygen atom, and
the blue nitrogen atom. The blue sticks show the nitrogen atom in
stick form.
Y. Cai et al. A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3581
found to be in close contact with the exocyclic substi-

tuent of d-p-HPH. Among these residues, Tyr153 is
well conserved, and previous investigation has revealed
that this tyrosine plays a very important role in coordi-
nating the substrate by forming a hydrogen bond with
the 4O of the hydantoinic ring [21,27]. Therefore,
Phe63, Leu92 and Phe150 were chosen for mutagenesis
analysis in order to identify the functional role of these
residues in the active center.
Initially, all three residues were mutated to Ala (a
smaller hydrophobic residue) individually, with the
hypothesis that this will enlarge the substrate binding
pocket in the neighborhood of the exocyclic substitu-
ent of the substrate. However, the results showed that,
in contrast to our expectations, all three mutations led
to a drastic decrease of HYD
Js
activity (data not
shown). It was therefore assumed that, for better per-
formance of the enzyme, a binding pocket of appropri-
ate size is necessary. We then replaced the three
residues with a range of amino acids using site-directed
saturated mutagenesis, and the activity of all the
mutants was measured (Fig. 6). The results showed
that the enzyme lost its activity dramatically when
Phe63 was mutated to any charged residues, although
positively charged residues (Lys and Arg) seemed to
have less effect than negatively charged ones (Glu and
Asp), while mutation of Phe63 into other amino acids
allowed the enzyme to retain similar activity. Leu92 is
one of the major constituents of the hydrophobic lids

of the substrate binding pocket. Replacement of Leu92
by polar and ⁄ or charged residues led to a serious
decrease of enzyme activity. According to our docking
model, Phe150 formed a close contact with the exocy-
clic group of the substrate. When Phe150 of the wild-
type enzyme was mutated into any other residue, the
enzyme lost nearly all its activity.
To further verify the importance of hydrophobic res-
idues in the SGLs, another residue in SGL3, Leu157,
was also chosen for site-directed mutagenesis analysis.
We substituted Leu157 by Asp, Ala, Ile or Val resi-
dues, and then checked the enzyme activity. The
results again showed that the hydrophobicity of SGLs
was important to retain enzyme activity. When Leu157
was mutated to Asp, a charged residue, the enzyme
lost its activity completely, while mutagenesis of
Leu157 to one of the other three residues retained
enzyme activity to varying degrees (Table 4).
Functional expression of HYD
Js
by co-expression
of chaperone GroEL/S
As shown in Fig. 2, overexpression of HYD
Js
under
the control of the T7 lac promoter resulted in protein
aggregation in E. coli. However, it has been extensively
reported that co-expression of a molecular chaperone
can alleviate this phenomenon [29,30]. To help HYD
Js

fold properly, we used the Takara chaperone plasmid
system to co-express HYD
Js
. The results showed that
construct pGro7, which expresses GroES–GroEL, can
improve soluble expression of HYD
Js
(Fig. 7), but
other chaperones tested did not assist the heterolo-
gously expressed HYD
Js
to fold properly (data not
shown), as analyzed by SDS–PAGE [31]. To confirm
this, whole-cell conversion of d,l-p-HPH was also
WT
F63C
F63D
F63E
F63G
F63H
F63I
F63K
F63L
F63M
F63N
F63P
F63Q
F63R
F63S
F63T

F63V
F63W
F63Y
WT
L92C
L92D
L92E
L92F
L92G
L92H
L92I
L92K
L92M
L92N
L92P
L92Q
L92R
L92S
L92T
L92V
L92W
L92Y
WT
F150C
F150D
F150E
F150G
F150H
F150I
F150K

F150L
F150M
F150N
F150P
F150Q
F150R
F150S
F150T
F150V
F150W
F150Y
120
100
80
60
Relative activity (%)
40
20
0
120
100
80
60
Relative activity (%)
40
20
0
100
80
60

Relative activity (%)
40
20
0
F63
L92
F150
Fig. 6. Relative activities of mutations at Phe63, Leu92 and
Phe150 of HYD
Js
. The mutated genes at the three sites were
expressed equally well as determined by SDS–PAGE (data not
shown). The activity was determined by measurement of 1 mL of
each mutant culture (22 °C, 48 h in LB medium).
A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1 Y. Cai et al.
3582
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS
performed. The activity analysis results showed that
co-expression with GroES–GroEL increased the
whole-cell activity approximately threefold (Table 5).
Discussion
Hydantoinase activity has been found in a wide spec-
trum of microorganisms, such as the genera Arthrob-
acter, Pseudomonas, Bacillus and Flavobacterium [32].
The conventional method of isolating new hydantoin-
ases involves direct screening of likely bacteria strains
for desired activity. However, as complete genome
sequences are now available for a number of microor-
ganisms, a new approach has arisen to identify puta-

tive target enzymes by coupling genomics database
mining with activity screening [1]. In this study, a new
hydantoinase from the Jannaschia sp. CCS1 genome,
designated HYD
Js
, was successfully identified using
this approach. Biochemical analysis showed that the
specific activity of this enzyme is approximately three
times higher than that of HYD
Bp
when using d,l-p-
HPH as the substrate. The study demonstrated that,
by coupling activity screening with genomics database
mining, the efficiency of discovering new enzymes for
industrial applications can be improved.
The 3D structures of several hydantoinases have
been published to date [21,27,33,34]. Analyses of the
3D structures could shed light on the relationships
between structure and function, and may help directed
evolution to further improve the catalytic activity. As
one example, Cheon et al. (2003) successfully improved
the catalytic properties of a d-hydantoinase by site-
directed and ⁄ or saturation mutagenesis based on anal-
ysis of its 3D structure [35,36]. If no crystal structure
is available, homology modeling is a powerful tool to
investigate the structure–function relationship. Based
on the homology model constructed in this study, we
were able to infer the possible reasons for high cata-
lytic activity in HYD
Js

. The highly conserved histidine
residues H56, H58, H181 and H237 were found to be
involved in metal binding [25], while the SGLs consti-
tute the substrate binding pocket. In particular, the
residues Phe63, Phe150 and Tyr153 formed close con-
tacts with the exocyclic substituent of the substrate.
These residues could be important for the substrate
specificity. It has been proposed that the size of the
substrate binding pocket and the hydrophobicity of
the residues near the exocyclic substituent of the sub-
strate play an important role in d-hydantoinase activ-
ity [35]. Superimposition of the structures of HYD
Js
and HYD
Bp
revealed that there is a distance of 2.9 A
˚
between the positions of the Ca of Ala156 in HYD
Js
and the Ca of Met156 in HYD
Bp
located in the SGL3,
which leads to the increased size of the substrate bind-
ing pocket of HYD
Js
. The side chain of Ala156 is
much smaller than that of Met156, which could fur-
ther increase the size of the substrate binding pocket.
In HYD
Js

, the sizes of the substrate binding pocket
and the entrance to the active site are increased com-
pared to those of HYD
Bp
, making it more accessible
for large substrates (Fig. 5). Our study provided
another indication that a enlarged substrate pocket
may be responsible for increased catalytic activity in
d-hydantoinases.
Fig. 7. Effects of co-expression of GroEL–GroES and HYD
Js
on sol-
uble expression of HYD. Strains expressing HYD
Js
harboring (+) or
not harboring ()) plasmid pGro7 were tested with induction of
GroEL–GroES (+) or without induction ()). tot, total proteins; ppt,
precipitate fraction; sup, supernatant fraction. Lane M, molecular
weight standard. The arrow indicates expression of GroEL.
Table 5. Relative activity of whole cells co-expressing HYD
Js
with
GroEL–GroES towards
D,L-p-HPH. The indication of pGro7 and L-ara
were the same as Fig. 7.
pGro7
L-ara Relative activity for D,L-p-HPH (%)
))100
) + 118
+ ) 108

+ + 317
Table 4. Site-directed mutagenesis analysis of Leu157, using D,L-p-
HPH as the substrate. The relative activity for the various mutants
is shown as a percentage of the activity of wild-type HYD
Js
for this
substrate.
Mutation Relative activity (%)
Wild-type 100
L157D 11.0
L157A 33.6
L157I 94.0
L157V 98.3
Y. Cai et al. A novel high-activity
D-hydantoinase from Jannaschia sp. CCS1
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3583
In HYD
Js
, the hydrophobic interactions within the
three SGLs in the substrate binding pocket seem to
be important for the substrate specificity, as observed
for other hydantoinases [27,35,36]. This is generally
true for HYD
Js
, for example mutation of Leu92 to
highly hydrophobic residues (i.e. Ala, Ile, Val and
Phe) retains the enzyme activity. However, mutation
of Phe63 to uncharged or even neutral residues can
retain HYD
Js

activity, and intriguingly, mutagenesis
of Leu92 to Ala and Val, two smaller hydrophobic
residues, actually reduced the catalytic activity to less
than approximately 50% of that of the wild-type
enzyme. In addition, replacement of Leu92 by Ile or
Phe had a negligible effect on the enzyme activity.
These results imply that, while a hydrophobic envi-
ronment is important for the binding pocket, an
appropriate side-chain size might also be important
for the activity. It is speculated that a smaller side
chain at this position (position 92) might have an
effect on the 3D structure of the hydrophobic lid
formed by SGL2, further decreasing the enzyme
activity. Mutagenesis analysis of Leu157 led to the
same conclusion. Phe150 is a very important residue
that is also highly conserved among all hydan-
toinases. The aromatic group of the Phe150 residue is
located in the vicinity of the exocyclic substituent of
the substrate. Although mutagenesis of Phe150 into
other residues caused nearly complete activity loss,
replacement of Phe150 by Tyr retained about 20% of
HYD
Js
activity. This suggests that the hydrophobic
interaction of Phe150 with the exocyclic group of
substrate may be critical for the catalytic activity.
The results again demonstrate that hydrophobicity
of the substrate binding pocket is necessary for the
catalytic activity, even though there may be other
requirements for residues at other positions, such as

side-chain size or polarity. Better understanding of
the roles of each of these residues will enable manipu-
lation of these SGLs by rational design or molecular
evolution methods to obtain the desired catalytic
activity.
Overexpression of heterologous proteins often results
in the formation of inclusion bodies in E. coli.
Co-expression of molecular chaperones is an easy way
to help heterologous proteins fold in the right way
[29,30]. It has been reported previously that soluble
expression of d-hydantoinase and carbamoylase can be
improved by co-expression with the molecular chaper-
ones DnaJ–DnaK and GroEL–GroES, respectively
[37]. In the case of HYD
Js
expression in E. coli,
GroEL–GroES was found to increase the soluble
expression of HYD
Js
remarkably; however, no effect
on HYD
Js
soluble expression was found by co-express-
ing DnaJ–DnaK. Complete conversion of d,l-p-HPH
requires the activities of both hydantoinase and DCase
in a two-step process, and we have previously found
that co-expression of GroEL–GroES can also improve
the soluble expression of DCase [31], which confers
more application advantages to HYD
Js

as a single set
of chaperones can assist soluble expression of both
HYD
Js
and DCase.
Although almost all hydantoinases that are currently
applied in industry were obtained from microbial
sources, the exact metabolic function and natural sub-
strates of hydantoinases in microbes are still far from
clear. However, a catalytic mechanism for their counter-
part in eukaryotes, dihydropyrimidinases, has been
proposed [38]. In eukaryotes, the enzymes catalyze
opening of the ring of 5,6-dihydrouracil to produce
N-carbamyl-b-alanine and of 5,6-dihydrothymine to
produce N-carbamyl-b-amino isobutyrate, which repre-
sents the second step in the three-step reductive degrada-
tion pathway of uracil, thymine and several anti-cancer
drugs [38]. Interestingly, annotation of the DNA
sequences flanking the Jannaschia sp. CCS1 HYD
Js
revealed an ORF encoding a putative allantoate amido-
hydrolase, which is part of the urate catabolic pathway
in many organisms [8]. In fact, by genome data mining,
another hydantoinase (HYD) was also found in the
Jannaschia sp. CCS1 genome besides HYD
Js
. However,
in contrast to HYD
Js
, the second HYD was not able to

hydrolyze d,l-p-HPH (data not shown), and no nucleo-
base metabolic gene was found near the second hyd
gene. Although genetic and biochemical studies are still
required to elucidate the in vivo function of HYD
Js
,itis
speculative that HYD
Js
might also be involved in the
degradation pathway of pyrimidines in Jannaschia sp.
CCS1. This speculation is supported by the fact that
HYD
Js
presents much higher activity towards DHU
than towards other substrates.
In conclusion, by combining genome database min-
ing and activity screening, we have successfully identi-
fied a new d-hydantoinase, HYD
Js
, from Jannaschia
sp. CCS1, which has a three times higher specific
activity than HYD
Bp
, the most widely used d-hydan-
toinase in industry. Biochemical characterization and
structural analysis of HYD
Js
suggested that the
enlarged substrate binding pocket could contribute to
its higher activity, allowing easy access to the cata-

lytic center and a higher turnover rate of the sub-
strate. While the information obtained in this study is
also important with regard to the continuous efforts
to improve HYD activity by a protein engineering
approach, the high activity of HYD
Js
makes it a
potentially useful enzyme for production of d-p-HPG
on an industrial scale.
A novel high-activity D-hydantoinase from Jannaschia sp. CCS1 Y. Cai et al.
3584
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS
Experimental procedures
Genome mining and identification of putative
D-hydantoinase genes
Using the amino acid sequence of HYD
Bp
(AAL37185) as a
query, BLAST searches for homologous proteins were per-
formed against the NCBI genome database. The homology
hits with less than 70% identity were checked further against
the BRENDA enzyme database (nda-enzyme-
s.info, March 2005) based on a name search. Sequence align-
ment was performed using clustal w [39].
Cloning and expression of putative HYDs
The genomic DNA of Jannaschia sp. CCS1 and Pseudomo-
nas fluorescens PfO-1 were kindly supplied by Mary Moran
(University of Georgia, Athens, GA) and Stuart Levy (Tufts
University School of Medicine, Boston, MA), respectively.
The genomic DNA of Streptomyces coelicolor A3(2) was

isolated in our laboratory. The hyd genes were amplified
using gene-specific primers with BamHI and HindIII recogni-
tion sites. The resulting PCR fragments were purified and
double-digested using BamHI and HindIII, and cloned into
pET28a(+) to generate pHYDs. The pHYDs were intro-
duced into E. coli strain BL21(DE3) to allow gene expression
under the control of T7 lac promoter. Cells with the targeting
constructs were incubated at 37 °C with agitation to the mid-
log phase, and induced using isopropyl-b-d-thiogalacto-
pyranoside at a final concentration of 1 mm.
Bacterial strains and culture conditions
E. coli strain DH5a was used for the cloning and amplifica-
tion of all constructed plasmids. Overexpression of target
genes was performed in E. coli BL21(DE3) (Novagen,
Shanghai, China). All strains were grown and maintained
in LB medium or TB (1.2% tryptone, 2.4% yeast extract,
4& v ⁄ w glycerol, 0.17 m KH
2
PO
4
and 0.72 m K
2
HPO
4
).
Appropriate antibiotics were added into the medium during
strain cultivation when necessary. Cultures were incubated
at 22 °C for 48 h or at 37 °C for 24 h with agitation at
200 rpm unless otherwise stated.
Enzymatic activity assay and characterization

The assay for hydantoinase activity was performed at 40 °C
with constant shaking. The reaction mixture contained
50 mm Tris ⁄ HCl pH 8.0, 1% w ⁄ v d,l-p-HPH and the
enzyme. After shaking at 150 rpm for 30 min, the reaction
was stopped by adding an equal volume of 1.0 m HCl to
the reaction mixture. The amount of product formed in the
supernatant of the reaction mixture was determined by
HPLC at 229 nm (1100 series, Agilent Technologies,
Shanghai, China). The HPLC system was equipped with a
ZORBAX Eclipse XDB-C8 column (internal diameter
4.6 mm, length 150 mm, Agilent Technologies). The mobile
phase used was 12% v ⁄ v methanol and 0.39& v ⁄ v acetic
acid. The flow rate was set at 1 mLÆmin
)1
. One unit of
enzyme activity was defined as the amount of enzyme
required to produce 1 lmol product per minute under the
conditions stated above.
Protein purification and analysis
The heterologously expressed proteins were purified using a
Ni
2+
affinity column (Ni-Sepharose high-performance, GE
Healthcare, Shanghai, China). After induction for 10 h,
cells expressing target proteins were harvested from 100 mL
culture medium by centrifugation at 4000 g and 4 °C for
10 min, and resuspended in 10 mL lysis buffer (50 mm
Tris ⁄ HCl pH 8.0, 300 mm NaCl, 10 mm imidazole). The
suspension was sonicated at 4 °C (5 s impulse and 20 s
break, 30 cycles). The lysates were centrifuged at 15 000 g

for 20 min at 4 °C. Supernatant fractions of the lysates
were mixed thoroughly with 1 mL of Ni-Sepharose medium
on ice by shaking, and applied to a 5 mL column. Wash
buffer (25 mL; 50 mm Tris ⁄ HCl pH 8.0, 300 mm NaCl,
50 mm imidazole) was used to elute non-specific binding
proteins. The target proteins were finally eluted using elu-
tion buffer (50 mm Tris ⁄ HCl pH 8.0, 300 mm NaCl,
500 mm imidazole). All steps were performed at 4 °Cto
minimize proteolysis of target proteins. The purified pro-
teins were dialyzed against a buffer containing 50 mm
Tris ⁄ HCl pH 8.0, in order to eliminate imidazole, and then
stored at 4 °C for further use. Protein purity was estimated
by SDS–PAGE stained with Coomassie brilliant blue [40].
Protein concentration was determined by the Bradford
method using bovine serum albumin as a standard [41].
The native molecular mass of recombinant HYD
Js
was
determined by gel filtration on a Superdex 200 10 ⁄ 300 GL
column (GE Healthcare) that had been previously cali-
brated using standard molecular weight proteins. The flow
rate was set to 0.8 mL Æmin
)1
. The subunit molecular mass
was also estimated by SDS–PAGE.
Characterization and comparative analyses of
HYD
Js
and HYD
Bp

The optimal temperature for activity of HYD
Js
with d-p-
HPH as substrate was determined by measuring the reaction
at a series of temperatures ranging from 30 to 70 °Cin
50 mm Tris ⁄ Cl pH 8.0. The optimal pH of HYD
Js
was deter-
mined at 40 °Cin50mm phosphate buffer (pH 6.0–8.0),
50 mm Tris ⁄ HCl (pH 7.0–9.0) or 50 mm glycine ⁄ NaOH (pH
9.0–10.0). Kinetic parameters were determined as described
previously [42]. HYD
Bp
from B. pickettii was used as a con-
trol for comparison of kinetic parameters. The substrate
specificity of HYD
Js
was determined under the conditions
stated above using dihydrouracil (DHU), hydantoin,
Y. Cai et al. A novel high-activity D-hydantoinase from Jannaschia sp. CCS1
FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3585
d,l-p-HPH, dimethylhydantoin, phenylhydantoin, diphenyl-
hydantoin, 5-(hydroxymethyl)uracil, benzylhydantoin and
isopropylhydantoin as the substrates. Product detection was
performed by colormetric assay using p-dimethylaminobenz-
aldehyde as the color reagent [19].
Structural analysis by comparative-modeling
techniques
The homology model of HYD
Js

was constructed using
SWISS-MODEL [43] with the structure of D. discoideum
dihydropyrimidinase [33] as the specific template. The crystal
structure was downloaded from PDB (accession number
2FTW) with a resolution of 2.0 A
˚
by X-ray diffraction. The
template of dihydropyrimidinase from D. discoideum 2FTW
was selected as a template by a BLAST search, as it has the
highest sequence identity to HYD
Js
of all available crystal
structures. The coordinates for zinc atoms and the water
molecule in the active site were defined according to the
position in the template D. discoideum dihydropyrimidinase
2FTW by structural superimposition. The quality of the
homology model HYD
Js
was checked by ProSA [26].
Alignments for sequence analysis were performed using
T-COFFEE [44].
Docking simulation of substrate on the active site
of HYD
Js
The substrate binding pockets of HYD
Js
and the mode of
interaction between substrate and enzyme were analyzed
by substrate docking in the active site using AutoDock4
[45]. Enzyme structures as the receptor were prepared for

docking in AutoDock4. For preparation of the ligands,
all structures were converted from isomeric SMILES
(Simplified Molecular Input Line Entry System) to 3D
structures using CORINA [46]. Partial charges of the
ligands were calculated using PETRA [47] (http://www2.
chemie.uni-erlangen.de/services/petra/smiles.phtml). Zn
2+
parameters were used as described previously [48]. The
grid map was created by AutoGrid4 with the grid center
at zinc I, grid size 40, and spacing 0.375. The docking in
AutoDock4 was performed using default values. The
bonded substrates were selected according to the orienta-
tion known from E. coli dihydroorotase (PDB accession
number 2E25) [49].
Site-directed mutagenesis of HYD
Js
Site-directed saturated mutagenesis for wild-type HYD
Js
at
three mutation sites, Phe63, Leu92 and Phe150, was
performed by in vitro mutagenesis using double-stranded
DNA templates [26]. Plasmid pHYD
Js
was used as the
template. In addition, site-directed mutagenesis of Leu157
was performed in a similar way. All the mutations were
confirmed by DNA sequencing. Expression of each and all
of the mutants in E. coli was checked by SDS–PAGE.
Co-expression of HYD
Js

with chaperone
molecules GroEL–GroES
The chaperone construct pGro7 was purchased from Taka-
ra Bio Inc. (Dalian, China), and then transformed into the
E. coli BL21(DE3) strain to generate E. coli BL21(DE3) ⁄
pGro. The HYD
Js
expression construct pHYD
Js
was also
transformed into E. coli BL21(DE3) ⁄ pGro to generate
co-expression strain E. coli BL21(DE3) ⁄ pGro ⁄ pHYD
Js
.
The co-expression strain was inoculated into LB medium
containing 17 lgÆmL
)1
chloramphenicol and 3 mgÆmL
)1
l-arabinose for induction of GroEL–GroES. After incuba-
tion at 37 °C until the cells reached an A
600
of 0.6, the cells
were induced with 0.5 mm isopropyl-b-d-thiogalactopyrano-
side and cultured at 30 °C for another 10 h to allow expres-
sion of the target gene.
Acknowledgements
We thank Mary Moran (University of Georgia, Athens,
GA) and Stuart Levy (Tufts University School of Medi-
cine, Boston, MA) for genomic DNA of Jannaschia sp.

CCS1 and Pseudomonas fluorescens PfO-1, respectively.
This work was supported by the International Scientific
Collaboration Program of Shanghai (grant number
075407065), the Knowledge Innovation Program of the
Chinese Academy of Sciences (KSCX2-YW-G-018,
KSCX2-YW-G-049), the National High-tech Research
and Development Program of China (2007AA02Z205),
the Knowledge Innovation Program of Shanghai Insti-
tute for Biological Sciences, Chinese Academy of
Sciences (2007KIP102), and the National Basic
Research Program of China (2007CB707803).
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