Molecular insights into substrate specificity of Rhodococcus ruber CGMCC3090 by gene cloning and homology modeling

Molecular insights into substrate specificity of Rhodococcus ruber CGMCC3090 by gene cloning and homology modeling

Enzyme and Microbial Technology 52 (2013) 111–117 Contents lists available at SciVerse ScienceDirect Enzyme and Microbial Technology journal homepag...

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Enzyme and Microbial Technology 52 (2013) 111–117

Contents lists available at SciVerse ScienceDirect

Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt

Molecular insights into substrate specificity of Rhodococcus ruber CGMCC3090 by gene cloning and homology modeling Shiwei Wang a,b , Yujie Dai a , Jianxin Wang a , Yanbing Shen a , Ying Zhai a , Heng Zheng c , Min Wang a,∗ a Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science and Technology), Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China b Colleges of Life Sciences and Engineering of Qiqihar University, Qiqihar 161006, PR China c Schools of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China

a r t i c l e

i n f o

Article history: Received 14 June 2012 Received in revised form 10 November 2012 Accepted 12 November 2012 Keywords: Rhodococcus ruber NHase Structure Function Homology modeling Docking

a b s t r a c t The primary aim of this study was to decipher the catalytic functions of the NHase with wide substrate spectra from Rhodococcus ruber CGMCC3090 by computer modeling and substrate docking. 3D structure model of the enzyme was built by computer modeling to obtain the optimal structure. The larger binding site cavity (559 A˚ 3 ) indicated that this NHase may catalyze a large variety of substrates of nitriles. Some key residues such as ␣Glu82, ␣Gln83, ␤Tyr71, ␤ Tyr72, ␤ Arg52 and ␤ Arg55 surrounding the binding site were unique compared with those of 3QXE as a template, indicating that the enzyme may have unusual substrate specificity. The docking and the biotransformation experiments demonstrated that the special docking pose and shorter distance proved to be more effective for the enzyme to improve function. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Nitrile hydratase (NHase) is a key enzyme in the bienzymatic pathway for the conversion of nitriles into amides [1]. A number of microorganisms-producing NHase were isolated and some enzymes were purified and characterized. The NHases have a wide range of physicochemical properties and substrate specificities [2]. They generally are composed of two types of subunits (␣ and ␤) complexes in varying numbers. These NHases are usually metalloenzymes containing either cobalt or iron. On the basis of the metal ion present, NHases can be classified into two groups: ferric NHases and cobalt NHases. The application of NHases has gained attention and also experienced industrialization globally recently. For example, large NHase plants [3] were set up in Japan (Mitsubishi Rayon Co., Ltd), France (SNF Floerger) and China (Lonza Guangzhou Fine Chemicals). Though recent advances have broadened the scope of the potential applications of the versatile biocatalyst, further reaction mechanism-oriented studies are required to fully exploit their biotechnological potential in biotransformation, bioremediation and environmental protection.

∗ Corresponding author at: No. 29, 13th Avenue, TEDA, Tianjin 300457, PR China. Tel.: +86 02260601256; fax: +86 02260602298. E-mail address: [email protected] (M. Wang). 0141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2012.11.004

The Rhodococcus ruber CGMCC30900 strain [4,5] was isolated from soil samples polluted by nitrile compounds. With high conversion capability, broad substrate spectra, the bacterium of R. ruber CGMCC30900 has demonstrated its wide application potential in biotransformation and organic synthesis. Though the Rhodococcus spp. have been demonstrated to possess either a bienzymatic system (nitrile hydratase and amidase) or a trienzymatic system [6], for NHase from R. ruber CGMCC3090 [7], interestingly, all the nitriles tested were hydrated to (cyano-) amides as the main products; almost no amidase or nitrilase activity was observed in the cells. NHase is a promising protein for rational design of biocatalyst, but the molecular details of the catalytic activity of NHases are not known comprehensively [8]. In all known NHases, the active site [9–11] exhibits the common motif: Cys-X-Y-Csd-SerCea, where Csd and Cea are posttranslationally modified cysteine residues. There are three possible catalytic mechanisms [12] proposed to date, but the solved structure of NHases contained neither nitrile nor amide molecule. Although with the advances in X-ray crystallography, many protein structures have been determined with high resolution, these static crystal structures need to be reevaluated with more comprehensive methods, such as biochemical assay, in order to explain the functions of dynamic proteins. At present, the structure of active enzymatic site of NHases has been defined, but a number of opening questions need to be addressed in order to understand this enzyme better and to facilitate its industrial applications. Among these questions, substrate specificity and

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regioselectivity are still important issues. Different NHases are specific for certain nitrile substrates. Therefore, explaining the possible intrinsic reason that why some substrates can be catalyzed specifically has become our priority. In this paper, the study is aimed at addressing the relationship between structure and enzymatic activity of R. ruber CGMCC3090 NHase by homology modeling and substrate docking. The amino acid sequence of NHase from R. ruber CGMCC3090 was determined by gene cloning and its 3D structure was built by homology modeling. The cavity volume and key amino acids of NHase model were compared with the template of 3QXE for further enunciating and deciphering the possible reaction mechanism of the NHase from R. ruber CGMCC3090. In order to test the feasibility of constructed structure model, docking experiments with substrates was carried out. Biotransformation experiments were also performed for studying and analyzing intrinsic reasons why different substrates can be docked with the NHase successfully. 2. Materials and methods 2.1. Chemicals and strain The PCR primers, Taq DNA polymerase and pCRTOPO2.1 were obtained from the Sangon (Shanghai, China). DNA markers and other reagents were purchased from Mycomebio (Beijing) Bio-medical Science & Technology Center. Substrates containing acrylonitrile, adiponitrile, 3-cyanopyridine, phthalonitrile were purchased from Sigma–Aldrich (USA). All chemical solvents and salts used were of analytical grade or higher. The R. ruber CGMCC3090 strain was obtained from the China General Microbiological Culture Collection Center. 2.2. Preparation of seed culture Agar medium and seed culture medium were prepared according to the methods [13] reported previously. Glycerol stock of bacteria of R. ruber CGMCC3090 (100 ␮L) were plated onto agar medium and cultured at 28 ◦ C for 1–3 days to obtained single colonies. A single colony was streaked on the agar medium plate and cultured aerobically at 28 ◦ C for 3–4 days. The fresh bacterial lawn was sub-cultured into the liquid seed media (50 mL) in a 250 mL Erlenmeyer flask and agitated at 180 rpm on a rotary platform at 28 ◦ C for 72 h. A maximum density of ca. 5 × 108 cells mL−1 was estimated at its logarithmic phase. 2.3. Cloning of NHase gene The genome DNA of the R. ruber CGMCC3090 strain was prepared as previously described [14]. Sample of DNA with OD260 1.8 was obtained and used as the template for the polymerase chain reaction. In order to clone the NHase gene, two pairs of primers targeting at the RD and DR regions were chosen. Degenerate primers were designed using other reported NHase sequence [15]. The upstream primer is RDCo225 (CCG GCC GGT AVA GCC GAC C), the downstream primers are DRCol711 (CTN CCT GCG GTG TGA GCG) and DRCol804 (CGG TGA ATA ACA AGC TCG CC) respectively. The polymerase chain reaction was carried out using the protocol reported previously [15] . The PCR products were verified by electrophoresis of aliquots of PCR mixtures (5 ␮L) in 1.8% agarose in 1 × TAE buffer and stained with ethidium bromide. 2.4. Sequencing and blasting PCR products were ligated into the pCRTOPO2.1 TA cloning vector. Positive colonies were sub-cultured and submitted to the Sangon Biotech Ltd for sequencing. The amino acid sequences deduced were submitted to NCBI GenBank and BLAST in RCSB protein data bank to analyze identity with the protein of the known NHases by blasting. 2.5. Molecular modeling The blast of protein amino acid sequences of ␣ and ␤ subunits of the nitrile hydratase from R. ruber CGMCC3090 in RCSB protein data bank was conducted using Accelrys Discovery Studio 2.1(DS 2.1). The crystal structure that has the highest sequence similarity to the nitrile hydratase from R. ruber CGMCC3090 in protein sequence was used as the model structure. The obtained 3D structure of the nitrile hydratase from R. ruber CGMCC3090 was further optimized with the molecular dynamics using CHARMm field in 0.145 M sodium chloride solution. The solved nitrile hydratase was first energy minimized using Smart Minimizer algorithm with Max Steps of 200 and RMS gradient of 0.1, then it was equilibrated for 10000 ps repeatedly with time step of 0.001 under constant pressure of 1.0 atm and 300 K of final temperature until the potential energy of the system became constant. The final structure of NHase from R. ruber CGMCC3090 was compared with that of the template selected.

2.6. Substrate docking The docking process was carried out using CDOCKER protocol [16]. Some molecule structures of substrates such as acrylonitrile, adiponitrile, nicotinonitrile (3-cyanopyridine), phthalonitrile etc. were built using the build fragment tool. All the operations above were carried out using D.S 2.1 package. The surface of the cavities of the catalysis center (binding site) were made in two steps. The cavities were first filled using the De Novo receptor protocol with the fragments in fragment library in DS 2.1 and then the solvent surfaces were added using the probe radius of 1.0. The cavity sizes were determined by the surface sizes. 2.7. Bio-transformation of the docked substrates The resting cells were used to hydrate the substrates screened by docking in order to verify the catalytic action of the NHase from R. ruber CGMCC3090 [13]. Activity of the NHase was assayed in a 1.5 mL reaction mixture consisted of bacteria liquid (100 ␮L) and different substrates (0.75 M) in 25 ␮M PBS buffer. The mixture was incubated at 28 ◦ C for 15 min and terminated by adding 10 ␮L 6 M hydrochloric acid. The reaction mixture was diluted and filtered through 0.22 ␮m filters. Different product concentrations were analyzed and Km and Vmax were calculated using the gas chromatography (GC) [17] or the high performance liquid chromatography (HPLC) method [18] as reported previously.

3. Results and discussion 3.1. Evaluation of PCR products and analysis of gene sequences The genomic DNA band extracted from the R. ruber CGMCC3090 strain was visualized in 1% agarose gel (data not shown). The target 1500 bp PCR product approximately was obtained using the primers (RDCo225 and DRCol804). The recombinants were sub-cultured and the NHase gene was sequenced. Results showed that nucleotide sequence of the PCR product consists of 1493 bases. The nearest distance with phylogenetic relationship was Nocard sp. JBRs (data not shown). It was reported [19] that the NHase genes of Rhodococcus can be divided into two types according to the relative position of the ␣-subunit and ␤-subunit. In type A, the ␣-subunit locates in the upstream of the ␤-subunit, while type B, per contra. Sequence alignment showed that the NHase gene of R. ruber CGMCC3090 is type B. 3.2. Blast of the deduced two subunits In order to obtain the structure of the NHase from R. ruber CGMCC3090, the amino acid sequences of ␤ and ␣ subunits were deduced and blasted in the RCSB Protein Data Bank. The ␤ subunit of the NHase consists of 229 amino acids (data not shown). The ␣ subunit has 203 amino acids. It can be seen from the blast result (Table 1) that the similarities of ␣ subunit are much higher than those of ␤ subunit. 3QXE B has the highest similarity when compared with NHase from R. ruber CGMCC3090. The identity was 37%. Meanwhile, 3QXE A and ␣ subunit have higher similarity with 57% identity. But when compared with ␣ chains of Fe-type NHases (PDB ID: 4FM4 A, 3A8G A, 2QDY A, 2ZCF A, 2AHJ A, 1AHJ A, 2D0Q A), the identity is only a value between 41% and 43%. Higher amino acid conservation was found within the ␣-subunit as compared to the ␤-subunit reflecting the important role of the ␣-subunit in the catalytic activity. The characteristic metal-binding site-CTLCSC (␣102-␣107) of NHases was found within the deduced amino acid sequence. Therefore these results further revealed that the NHase gene was present in the genome of R. ruber CGMCC3090, suggesting that NHase-producing strains are widely distributed among bacterial communities. It was reported that nitrile hydratases had a metal active center to which the nitrile group of the substrate bound, so the formation of nitrile hydratase was highly enhanced by the addition of ferrous (ferric ions) or cobalt ions into the medium [20,21]. The metal active center of NHase from R. ruber CGMCC3090 was identified as described previously [22]. Resting cells of R. ruber CGMCC3090

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Table 1 Protein blast results from protein data bank. PDB ID

Chain and type

Max score

Total score

Query coverage

Max identity

1UGQ A 1UGS A 1IRE A 1UGP A 1UGR A 1V29 A 3QXE A 3QZ5 A 2DPP A 3HHT A 1AHJ A 2D0Q A 2AHJ A 2QDY A 3A8G A 2ZCF A 2DD4 C 3QXE B 3QYG B 3QZ9 B 2DD5 C 3QYH B 4FM4 A

␣, Co ␣, Co ␣, Co ␣, Co ␣, Co ␣, Co ␣, Co ␣, Co ␣, Co ␣, Co ␣, Fe ␣, Fe ␣, Fe ␣, Fe ␣, Fe ␣, Fe C, Co ␤, Co ␤, Co ␤, Co C, Co ␤, Co ␣, Fe

241 239 233 233 232 227 223 222 219 219 179 174 170 170 169 168 164 157 156 155 155 153 152

241 239 233 233 232 227 223 222 219 219 179 174 170 170 169 168 164 157 156 155 155 153 152

0.43 0.43 0.43 0.43 0.43 0.45 0.42 0.42 0.46 0.46 0.45 0.42 0.42 0.45 0.45 0.42 0.43 0.52 0.52 0.52 0.43 0.52 0.42

0.59 0.58 0.58 0.58 0.57 0.56 0.57 0.56 0.55 0.55 0.43 0.45 0.44 0.42 0.42 0.44 0.45 0.37 0.37 0.37 0.44 0.37 0.41

exhibited no detectable conversion of acrylonitrile in cobalt-free medium for 48 h, and only when cobalt was added into medium, can the activity of NHase be detected. These phenomena indicate that the NHase from R. ruber CGMCC3090 is a metalloenzyme and belongs to the cobalt NHase family [7]. Based on the analysis above, the NHase from R. ruber CGMCC3090 was comprised of two different subunits, ␣- and ␤-subunits with cobalt ion as the prosthetic metal. 3.3. Homology modeling and possible catalytic functions The crystal structure of Co-type nitrile hydratase from P. putida (PDB ID: 3QXE), which has the highest ␤ subunit sequences similarity and a comparatively higher ␣ subunit sequence similarity to that of the nitrile hydratase from R. ruber CGMCC3090, was used as template to build homology model. The crystallization structure from ˚ The P. putida was obtained by X-Ray Diffraction (Resolution: 2.1 A). two cysteine residues of molecular structure of the nitrile hydratase from 3QXE (␣Cys115 and ␣Cys117) equatorially coordinated to the cobalt ion were post-translationally modified to cysteine sulfinic acid and cysteine sulfenate respectively [23,24]. The spatial structure of the NHase from R. ruber CGMCC3090 was built from the homology modeling protocol in Discovery Studio 2.1. Refine loops was selected as “true”. The core structure of the active center of Co3+ , ␣Cys102, ␣CSC (105–107) was directly copied from 3QXE [Cys112, CSC (115-117)] accordingly and the spatial structure of other residues was constructed by homology modeling protocol. In order to further optimize the homology modeling structure of NHase from R. ruber CGMCC3090, the molecular dynamics was conducted. The homology model was first energy minimized using the minimization protocol in DS 2.1 under 0.145 M/L sodium chloride aqueous solvent condition. The solvent was added by solvation protocol with the explicit periodic boundary model. Then the dynamics equilibration was carried out repeatedly with the constant pressure of 1.0 atm and 300 K of temperature until the potential energy became constant. The residue of ␣Cys102, ␣Csd105, ␣Cea107, and ␣Ser106 were in harmonic restraint. The 3D structure comparisons of NHase from R. ruber CGMCC3090 with 3QXE are shown in Fig. 1. The enzyme from R. ruber CGMCC3090 is composed of two subunits (␣, ␤). The active site exhibits the common motif: Cys-X-Y-CsdSer-Cea, where Csd (␣Cys105-SO2H) and Cea (␣Cys107-SOH) are

Fig. 1. Comparison of ribbon representation between the NHase structure of R. ruber CGMCC3090 and the crystal structure of 3QXE. *The NHase from R. ruber CGMCC3090 is in red ribbon and 3QXE is in green ribbon. The residues of Cys112, Csd115, Cea117 and Ser116 from 3QXE are shown in stick and the residues of Cys102, Csd105, Cea107 and Ser106 from homology model of Nhase from R. ruber CGMCC3090 in ball and stick.

postranslationally modified. The noncorrin Co3+ metal ion lies in the non-standard active site. There are some significant differences between the 3D structure of NHase of R. ruber CGMCC3090 and 3QXE, such as the regions A–D. The cavity shapes of the binding site are also very different from that of NHase from P. putida (3QXE). The binding site of 3QXE likes an oval (the white region in Fig. 2A) with a volume of 206 (Å3 ) which are composed of Co3+ and the residues of ␣Cys112, ␣Csd115, ␣Cea117 and ␣Ser116; however, that of NHase of R. ruber CGMCC3090 (see Fig. 2B) likes a heart with a volume of 559 A˚ 3 .The key residues surrounding the bind˚ are ␣Val92, ␣Gln93, ␣Cys112, ␣Csd115, ing site in 3QXE (near 4 A) ␣Ser116, ␣Cea117, ␣Tyr118, ␣Trp120, ␣Pro126 and ␣Arg170 in ␣ chain, while ␤Leu37, ␤Leu41, ␤Leu48, ␤Trp72 ␤Phe51, ␤Arg52, ␤Ile55 and ␤Tyr68 in ␤ chain, while the key residues surrounding the binding site in NHase from R. ruber CGMCC3090 are ␣Glu82, ␣Gln83, ␣Cys102, ␣Csd105, ␣Ser106, ␣Cea107and ␣Trp110 in ␣ chain, ␤Tyr71, ␤Tyr72, ␤His74, ␤Arg52, ␤Arg55 and ␤Ser51 in ␤ chain. From the spatial comparison of the two nitrile hydratases, we can see that Leu37, Leu41, Phe 46 and Phe 51 are stretching into the cavity of NHase of R. ruber CGMCC3090, which leads to the formation of smaller cavity of NHase from P. putida (3QXE) (see Fig. 3). These differences in structure may lead to significant diversity in catalysis of the hydration of the nitrile groups. The larger binding site volume of NHase of R. ruber CGMCC3090 permits the molecules with higher masses to be entered easily. Perhaps this is an important reason why the NHase have broad substrate spectra. Liya Song [25] reported that glutamine residue (␣Gln90) in the active center of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion. It may be useful for us to analyze the mechanism of biocatalysis of nitrile hydratase from R. ruber CGMCC3090 using the two amino acids (␣Glu82 and ␣Gln83) surrounding the binding site. It was

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Fig. 2. Cartoon representation of binding site of 3QXE and NHase from R. ruber CGMCC3090.

reported [26–29] that mutation of some conserved amino acids led to the loss of NHase activity, suggesting that ␤Arg, ␤Tyr and ␣Gln surrounding the binding site might stabilize the active center of NHase. The R. ruber CGMCC3090 NHase key amino acids such as ␤Tyr71, ␤Tyr72, ␤Arg52 and ␤Arg55 in ␤ chain surrounding the binding site can also play role in stabilizing the active center of NHase. Brodkin [30] used THEMATICS to identify residues in the second and third shells of the Co-type nitrile hydratase from P. putida (ppNHase) to improve further understanding of the roles of second- and third-shell residues in catalysis. Pseudomonas putida showed a diverse spectrum of metabolic activities, which was indicative of their adaptation to various niches, including the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. We presumed that three of the predicted second-shell residues (␣Asp155, ␤Glu56 and ␤His148) and one predicted third-shell residue (␤His74) also have similar effects on the catalytic efficiency of the enzyme from R. ruber CGMCC3090. A possible reason why the R. ruber CGMCC3090 strain is capable of catalyzing different nitriles is probably related to the special second- and third-shell residue of the R. ruber CGMCC3090 NHase. 3.4. Substrate docking and biotransformation In order to analyze the catalysis properties of the NHase from R. ruber CGMCC3090, several representative substrates were

Fig. 3. Amino acid residues of 3QXE stretching into the cavity of NHase of R. ruber CGMCC3090 (white surface).

chosen and used to dock into the binding site of NHase from R. ruber CGMCC3090 using the CDOCKER protocol in DS 2.1. The results showed that the aliphatic (di) nitrile, heterocyclic nitrile, aromatic di-nitrile substrates enter the binding site and is able to dock well in the active site (Fig. 4). The acrylonitrile lies upon Co metal atom, which is similar to the reported by Lukasz Peplowski [31]. However, adiponitrile, nicotinonitrile (3-cyanopyridine) and phthalonitrile are standing upon the cavity with nitrogen atom of CN directing to Co atom. The same case was also evidenced for Fe NHase [9]. On the other hand, the distances of docking are different. There are many factors influencing the enzyme catalysis activities, such as the suitable tunnel size for substrate entering the binding site, the binding energy between substrate and enzyme, and the binding energy difference between substrate and product molecules, etc. Besides all these mentioned above, the poses of the substrates and the distance between the reaction group in substrate and the active site might be the most intuitive. In order to verify the NHase catalytic functions and characteristics, biotransformation experiments were done using the R. ruber CGMCC3090 resting cells. The result of biotransformation of 3-cyanopyridine (the retention time: 2.530 min) to nicotinamide (the retention time: 4.408 min) by high performance liquid chromatography is shown in Fig. 5. Other results were shown in Table 2. Interestingly, although the selectivity to the substrates has no obvious change (data not shown), the specific conversion ratio to acrylonitrile is significantly higher than the other 3 substrates (adiponitrile, 3-cyanopyridine and phthalonitrile), suggesting that the docking poses of different nitrile substrates may be directly related to the specific conversion ratios. The docking pose lying upon Co metal atom may be more effective for the enzyme to improve catalytic properties than the pose standing upon the cavity with nitrogen atom of CN directing to Co catalysis atom. Moreover, the docking distance is an important factor that affects specific conversion ratio. Generally the catalytic site of NHase (Fe-/Co-ion center) is deeply buried in protein scaffold. Therefore substrate (nitrile molecules) must reach the NHase interior for catalysis and eventually product leave the catalytic cleft. The distance from NHase surface to Co3+ center is about 15 A˚ from one of the expected side [3]. As shown in ˚ The dockTable 2, all docking distance are far less than 15 A. ing distances also have certain relation to the specific conversion ratios. As far as the mono-nitriles like acrylonitrile (D = 4.458) and 3-cyanopyridine (D = 4.995) were concerned, the shorter the docking distance was, the higher the specific conversion was contrarily. The conclusion was also the same for the different dinitriles such as adiponitrile (D = 4.874) and phthalonitrile (D = 6.699). However, the R. ruber CGMCC3090 strain would rather catalyze mono-nitriles

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Fig. 4. Docking poses and distances of different nitriles ((A) acrylonitrile, (B) adiponitrile, (C) 3-cyanopyridine, (D) phthalonitrile) as substrates.

like acrylonitrile and 3-cyanopyridine than do dinitriles such as adiponitrile and phthalonitrile. Besides, the NHase has the highest Km (2.329 mol/L) and Vmax (0.246 mol/min/L) to acrylonitrile, suggesting that this substrate may be catalyzed easily. The analysis above shows that the catalytic function of the NHase is also clarified partly by docking screening and biotransformation assay.

Moreover, the docking screening may simplify the process of screening substrates. A ‘breathing’ and ‘flip-flop’ mechanism which might be a feasible hypothesis of the substrate binding mechanism have been proposed [32]. Nevertheless, gaining the more detailed insights for NHase from R. ruber CGMCC3090 needs further investigation.

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Table 2 The relationships among the docking pattern, the specific conversion ratio and two kinetic parameters (Km, Vmax). Classification

Substrate

Product

Docking posea

Docking distance

Specific conversion ratiob mM g−1 (dew)/min−1

Km (mol/L)

Vmax (mol/min/L)

Mono-nitriles

Acrylonitrile c 3-Cyanopyridine c Adiponitrile c Phthalonitrile c

Acrylamide Nicotinamide 5-Cyanovaleramide Orthocyano benzamide

A B B B

4.458 4.995 4.874 6.699

98.04 45.92 8.07 1.29

2.329 0.196 0.809 0.922

0.246 0.073 0.029 0.019

Dinitriles

a Differents docking poses: (A) is lying upon cobalt (Co) metal atom in the active site.; (B) is standing upon the cavity with the nitrogen atom of cyano (CN) directing to cobalt (Co) atom in the active site. b Specific conversion ratios on the basis of the initial substrate concentration and the final product concentrations were determined by high performance liquid chromatography (HPLC) or gas chromatography (GC).

c

The figure of the substrate used in the study is marked as follows: Acrylonitrile:

Adiponitrile:

; 3-cyanopyridine:

;

; Adiponitrile:

Acknowledgements The current work was supported by the National Natural Science Foundation of China (21076158, 21276196), the Program for New Century Excellent Talents in University (NCET-08-0911), the National High Technology Research and Development of China (2011AA02A211).

References

Fig. 5. HPLC spectrum of biotransformation of nicoinitrile.

4. Conclusions The significant different catalysis center shape and volume made the NHase have specific function, such as higher tolerance to acrylonitrile as substrate, better regioselectivity of di-nitrile. The larger binding site cavity indicating that the NHase had wide substrate spectra. The key residues responsible for hydration containing two amino acids (␣Glu82 and ␣Gln83) in ␣ chain and four amino acids (␤Tyr71, ␤Tyr72, ␤Arg52 and ␤Arg55) in ␤ chain surrounding the binding site are important for the NHase to stabilize the active center and further fulfill its especial catalytic task. Docking and biotransformation experiments showed that the enzyme have a preference for mono-aliphatic nitrile (acrylonitrile) rather than heterocyclic mono-nitriles (3-cyanopyridine), aliphatic dinitriles (adiponitrile) and aromatic dinitriles (phthalonitrile). Thus modeling and docking studies provide more supports not only for enunciating NHase mechanisms but also for substrate screening and biotransformation.

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