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: 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 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 [...]... d,l-5substituted hydantoins by an Agrobacterium tumefaciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity Biotechnol Lett 20, 707–711 Durr R, Vielhauer O, Burton SG, Cowan DA, Punal A, Brandao PFB, Bull AT & Syldatk C (2006) Distribu- A novel high-activity D-hydantoinase from Jannaschia sp CCS1 19 20 21 22 23 24 25 26 27 28 29 30 31 tion of hydantoinase activity... bacterial isolates from geographically distinct environmental sources J Mol Catal B Enzym 39, 160–165 Xu Z, Liu YQ, Yang YL, Jiang WH, Arnold E & Ding JP (2003) Crystal structure of d-hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability J Bacteriol 185, 4038–4049 Nanba H, Yajima K, Takano M, Yamada Y, Ikenaka Y & Takahashi... dihydropyrimidinase in the metabolism of some hydantoin and succinimide drugs Drug Metab Dispos 2, 103–112 Moller A, Syldatk C, Schulze M & Wagner F (1988) Stereo- and substrate-specificity of a d-hydantoinase and a d-N-carbamyl amino acid amidohydrolase of Arthrobacter crystallopoietes AM 2 Enzyme Microb Technol 10, 618–625 Ogawa J & Shimizu S (1997) Diversity and versatility of microbial hydantoin-transforming... enzymes J Mol Catal B Enzym 2, 163–176 Bommarius AS, Schwarm M & Drauz K (1998) Biocatalysis to amino acid-based chiral pharmaceuticals - examples and perspectives J Mol Catal B-Enzym 5, 1–11 Liljeblad A & Kanerva LT (2006) Biocatalysis as a profound tool in the preparation of highly enantiopure beta-amino acids Tetrahedron 62, 5831–5854 Kim GJ & Kim HS (1998) Identification of the structural similarity in.. .A novel high-activity D-hydantoinase from Jannaschia sp CCS1 Y Cai et al Experimental procedures Genome mining and identification of putative D-hydantoinase genes Using the amino acid sequence of HYDBp (AAL37185) as a query, BLAST searches for homologous proteins were performed against the NCBI genome database The homology hits with less than 70% identity were checked further against the BRENDA enzyme... concentration was determined by the Bradford method using bovine serum albumin as a standard [41] The native molecular mass of recombinant HYDJs was determined by gel filtration on a Superdex 200 10 ⁄ 300 GL column (GE Healthcare) that had been previously calibrated using standard molecular weight proteins The flow rate was set to 0.8 mLÆmin)1 The subunit molecular mass was also estimated by SDS–PAGE Characterization. .. the targeting constructs were incubated at 37 °C with agitation to the midlog phase, and induced using isopropyl-b-d-thiogalactopyranoside at a final concentration of 1 mm Bacterial strains and culture conditions E coli strain DH 5a was used for the cloning and amplification of all constructed plasmids Overexpression of target genes was performed in E coli BL21(DE3) (Novagen, Shanghai, China) All strains... the National High-tech Research and Development Program of China (2007AA02Z205), the Knowledge Innovation Program of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (2007KIP102), and the National Basic Research Program of China (2007CB707803) References 1 Altenbuchner J, Siemann-Herzberg M & Syldatk C (2001) Hydantoinases and related enzymes as biocatalysts for the synthesis of. .. Acknowledgements We thank Mary Moran (University of Georgia, Athens, GA) and Stuart Levy (Tufts University School of Medicine, 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,... and maintained in LB medium or TB (1.2% tryptone, 2.4% yeast extract, 4& v ⁄ w glycerol, 0.17 m KH2PO4 and 0.72 m K2HPO4) 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 . Isolation and molecular characterization of a novel D-hydantoinase from Jannaschia sp. CCS1 Yuanheng Cai 1 , Peter Trodler 2 , Shimin Jiang 1 ,. 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