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)

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

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-

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

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

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

(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

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

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-

.
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

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

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

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

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

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 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

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

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

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 (http://www.brenda-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

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

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

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

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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