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

By a process coupling genomic data mining with activity screening, a new hydantoinase, tentatively designated HYDJs, was identified from Jannaschia sp.. The specific activity of HYDJs on d

D -hydantoinase from Jannaschia sp CCS1

Yuanheng Cai1, Peter Trodler2, Shimin Jiang1, Weiwen Zhang3, Yan Wu1, Yinhua Lu1, Sheng Yang1 and Weihong Jiang1,4

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- or l-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 chiral compounds 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: whjiang@sibs.ac.cn

(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 HYDJs, was identified from Jannaschia sp CCS1 and overexpressed in Escherichia coli The specific activity of HYDJs on d,l-p-hydroxyphenylhydantoin as the substrate was three times higher than that of the hydantoinase originating from Burkholderia pickettii (HYDBp) 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 HYDJs were 7.6 and 50C respec-tively, similar to those of HYDBp Kinetic analysis showed that HYDJshas

a higher kcat value on d,l-p-hydroxyphenylhydantoin than HYDBp does Homology modeling and substrate docking analyses of HYDJsand HYDBp were performed, and the results revealed an enlarged substrate binding pocket in HYDJs, which may allow better access of substrates to the cata-lytic centre and could account for the increased specific activity of HYDJs Three amino acid residues critical for HYDJs activity, Phe63, Leu92 and Phe150 were also identified by substrate docking and site-directed muta-genesis Application of this high-specific activity HYDJscould 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.

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- or l-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

(HYDBp) and heterologously expressed in

Escherichi-a coli [19] The HYDBp 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

HYDBp was also determined, and its catalytic active

site was found to consist of two metal ions and six

highly conserved amino acid residues Although

HYDBp 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 HYDJs Biochemical analysis showed that HYDJshas a specific activity approximately three times higher than that of HYDBp 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 HYDJs, 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 engineersuggest-ing of the HYD for high catalytic activity In addition, the high specific activity HYDJs can be readily applied for industrial production of optically pure amino acids

Results

Genome database mining and identification of putativeD-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 HYDBp 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 HYDBp were eliminated to avoid repetitive characterization of enzymes similar to those previously identified, and the less homologous

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 HYDBp The strictly conserved residues are shaded black, and the residues relevant to metal ion binding are indicated by filled stars.

Fig 1 (Continued).

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 cenocepaciaAU 1054 (Q1BGK8),

Chloro-bium phaeobacteroides BS1 (Q4AGB4),

Desulfitobacte-rium hafnienseY51 (YP_518039), Jannaschia sp CCS1

(ABD54405), Polaromonas sp JS666 (Q12FP8),

Moo-rella thermoacetica ATCC 39073 (Q2RGZ6),

(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

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 (HYDJs) and

Pseu-domonas fluorescens PfO-1 (HYDPf) 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)⁄ pHYDJs and E coli BL21(DE3)⁄ pHYDPf

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 HYDBp was similar to that of other hydantoinases, which are mostly between 50 and

60 kDa [4] It is noteworthy that overexpression of HYDJsresulted in the formation of inclusion bodies in the precipitate fraction, which may lead to low activity

of whole-cell extract, while HYDBp and HYDPf were mainly expressed in soluble fraction under the experi-mental conditions used (Fig 3)

Purification and specific activities of HYDs HYDJs was purified to homogeneity from E coli BL21(DE3)⁄ pHYDJsby 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 HYDBp and HYDPf from

E coli BL21(DE3)⁄ pHYDBp and E coli BL21(DE3)⁄ pHYDPf was also performed Their specific activities for hydrolyzing d,l-p-HPH were also determined and compared The specific activity of HYDJs was about three times higher than that of HYDBp, and five times higher than that of HYDPf (Table 1) As the activity

of HYDPf at the whole-cell level was lower than that

of HYDBp, even though it seems to be more soluble than HYDBp(data not shown), we concluded that the specific activity of HYDPf may be less than that of HYDBp, 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.

characterization and evaluation of HYDJs from

Jannaschiasp CCS1

Characterization of HYDJs

To explore the possible cause of the higher specific

activity for conversion of d,l-p-HPH to

N-carbamoyl-p-hydroxyphenylglycine by HYDJs, the kinetic

parameters of HYDJs and HYDBp were comparatively

determined (Table 2) The mean Km values were

simi-lar for both enzymes, but HYDJs had a much higher

kcat, suggesting a higher turnover rate of HYDJs

compared to HYDBp

The pH and temperature dependence of HYDJs

activ-ity were measured (Fig 4) The results revealed an

opti-mal temperature of HYDJs of 50C for hydrolyzing

d,l-p-HPH, which is the same as that for HYDBp[19]

The optimal pH for the hydrolytic activity of HYDJs

was 7.6, slightly lower than that for HYDBp (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, HYDJshas an

advantage over HYDBp 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 HYDJs 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 HYDJsindicated a molecular mass of about 253 kDa, and, as the subunit molecular mass of the His-tagged recombinant HYDJswas estimated to be 56 kDa, these results suggest that HYDJs occurs as a homotetramer

in solution

Homology structural modeling of HYDJs

A homology model of the structure of HYDJs was generated to further investigate the structural basis for the higher activity of HYDJs compared to HYDBp The Z-score for the homology model HYDJs 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)

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 Km(m M ) kcat(s)1) kcat⁄ K m (m M )1Æs)1)

Fig 4 Temperature and pH dependence of HYDJs (A) Tempera-ture ⁄ activity profile of purified HYD Js ; (B) pH ⁄ activity profile of purified HYDJs.

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 HYDJs, 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 HYDJs and HYDBp (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 HYDJs, the size of the

substrate binding pocket and the entrance to the active

site are larger compared to those of HYDBp, making it

more accessible for larger substrates (Fig 5)

Docking analysis of substrate on the active site

of HYDJs

The substrate binding pocket in the active site was

inves-tigated based on the homology model to obtain more

information on substrate binding in HYDJs 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 HYDJs[28] to simulate the mode of

sub-strate binding The results suggest that the subsub-strate

binding pocket accommodates substrates with small side

chains better than those with large ones, which is in

accordance with the specific activity of HYDJsagainst

the tested substrates (Table 3)

Identification of active-site residues of HYDJs

On the basis of fitting d,l-p-HPH as a target substrate into the active site of HYDJs, 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 HYDJs, 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 HYDJs hydrolyzes dihydrouracil ND, enzyme activity

corre-sponding to less than 1% of the rate at which HYDJs hydrolyzes

dihydrouracil.

Substrates

Relative activity (%)

A

B

Fig 5 Homology model of HYDJs (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 HYDJsare 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.

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 HYDJs 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 HYDJsby co-expression

of chaperone GroEL/S

As shown in Fig 2, overexpression of HYDJs 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 HYDJs fold properly, we used the Takara chaperone plasmid system to co-express HYDJs The results showed that construct pGro7, which expresses GroES–GroEL, can improve soluble expression of HYDJs (Fig 7), but other chaperones tested did not assist the heterolo-gously expressed HYDJs 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

F63C F63D F63E F63G F63H F63I F63K F63L F63M F63N F63P F63Q F63R F63S F63T F63V F63W F63Y

L92C L92D L92E L92F L92G L92H L92I L92K L92M L92N L92P L92Q L92R L92S L92T L92V L92W L92Y

F150C F150D F150E F150G F150H F150I F150K F150L F150M F150N F150P F150Q F150R F150S F150T F150V F150W F150Y

120 100 80 60

40 20 0

120 100 80 60

40 20 0

100

80

60

e activity (%) 40

20

0

F63

L92

F150

Fig 6 Relative activities of mutations at Phe63, Leu92 and Phe150 of HYDJs 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).

performed The activity analysis results showed that

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 HYDJs, was successfully identified using this approach Biochemical analysis showed that the specific activity of this enzyme is approximately three times higher than that of HYDBp 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 HYDJs 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 HYDJs and HYDBp revealed that there is a distance of 2.9 A˚ between the positions of the Ca of Ala156 in HYDJs and the Ca of Met156 in HYDBplocated in the SGL3, which leads to the increased size of the substrate bind-ing pocket of HYDJs 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 HYDJs, the sizes of the substrate binding pocket and the entrance to the active site are increased com-pared to those of HYDBp, 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 HYDJson

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 (%)

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 HYDJsfor this

substrate.

In HYDJs, 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 HYDJs, 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 HYDJs 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

HYDJs 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 HYDJs expression in E coli,

GroEL–GroES was found to increase the soluble

expression of HYDJs remarkably; however, no effect

on HYDJssoluble 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 HYDJs as a single set

of chaperones can assist soluble expression of both HYDJsand 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 HYDJs 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 Jannaschiasp CCS1 genome besides HYDJs However,

in contrast to HYDJs, 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 HYDJs, it is speculative that HYDJs might also be involved in the degradation pathway of pyrimidines in Jannaschia sp CCS1 This speculation is supported by the fact that HYDJs presents much higher activity towards DHU than towards other substrates

In conclusion, by combining genome database min-ing and activity screenmin-ing, we have successfully identi-fied a new d-hydantoinase, HYDJs, from Jannaschia

sp CCS1, which has a three times higher specific activity than HYDBp, the most widely used d-hydan-toinase in industry Biochemical characterization and structural analysis of HYDJs 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 HYDJs makes it a potentially useful enzyme for production of d-p-HPG

on an industrial scale