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Infections caused by Staphylococcus species responsible for number of human and animal infections including arthritis, synovitis, metritis, osteomyelitis, vaginitis and spondylitis, which are diseases caused by Staphylococcus hyicus (SHL), known by Skin infections in horses, cattle, chickens, turkeys and pigs. The animal skin pathogen Staphylococcus hyicus is unique in having high A1 and minor A2 phospholipase activities, Micrococcus hyicus is commonly treated with penicillin, streptomycin, and tetracycline. However, the secondary effects caused during such treatment have aroused a need to develop a treatment based on lipase inhibition. No such lipase inhibitors for Micrococcus hyicus treatment are currently available. Thus, we have performed a docking study with the natural inhibitor, Crocetine using GOLD software. Our results have shown ten possible binding inhibitors to Staphylococcus hyicus, out of which one possibility was selected, based on the weakest interatomic distance of 1.3 Å. Therefore, we propose the selection and design of a potential inhibitor, Crocetine for the treatment of Micrococcus hyicus infections. However, this study has to be supported with in vitro and in vivo experiments to demonstrate the effectiveness of Crocetine in lipase inhibition.
Keyword: SHL, CROCETIN, LIPASE INHIBITION, DOCKING, GOLD.
Lipases (glycerol ester hydrolases, EC 126.96.36.199) catalyze the hydrolysis of ester bonds in long-chain acylglycerols. The Staphylococcus hyicus (S. hyicus) or Micrococcus hyicus lipase is the first lipase family I.6 (lipase classification scheme proposed by Arpigny and Jaeger, member of which a 3D-structure has been elucidated. The structure closely resembles that of two Geobacillus lipases from lipase family I.5, GSL and GSP, demonstrating the little difference between the two lipase families, the availability of large amounts of biochemical data and many crystal structures means that their catalytic mechanism, reaction selectivity and substrate specificity are very well understood. [1-4]. Twenty species of the families of staphylococci are currently identified, including the main species: Staphylococcus aureus, responsible for number human and animal infections. With the exception of a wound infection following the bite of a donkey, Staphylococcus hyicus has never been isolated from humans, Norwegian survey showed the absence of specific antibodies (antibodies anti-DNase) in 62 individuals. In healthy animals, Staphylococcus hyicus was isolated from the skin and nasal cavities of domestic or wild birds, of the skin, nasal cavities, tonsils and vagina of pig's skin and tonsils from cattle of the breast and milk of goats and skin of cats. This bacterium is responsible for infections in various animal species and, in many cases (skin infections of horses or cattle or pigs, metritis and vaginitis in pigs, the disease could be reproduced experimentally. In chickens and turkeys, Staphylococcus hyicus is responsible for arthritis, synovitis, osteomyelitis and spondylitis. This bacterium is often isolated in pure culture from skin lesions and various organs (liver, lungs). Staphylococcus hyicus was also isolated from respiratory lesions in canaries and a case of conjunctivitis has been described in one ostrich. In animals, especially horses Staphylococcus hyicus is the cause of various skin lesions, abscesses and can be isolated from lesions of dermatophilosis (Dermatophilus). In the cow and goats, Staphylococcus hyicus is responsible for sub-clinical mastitis and skin infections. In cats, the presence of Staphylococcus hyicus has been associated with skin lesions. Hence, lipase has been identified as a possible target for the development of novel anti-fungal therapeutic compounds . The Crocin is a carotenoid glycosylated and its metabolite resulting aglycone is known under the name of crocetin (Figure 2), the major constituents of the plant Gardenia jasminoids. Those are reversible competitive inhibitors of pancreatic lipase with an IC50 value of 28.63 mM for crocin. As well, both compounds were presented in different work activities hypotriglycéridiques and hypocholestéromiques . Most lipases display interfacial activation, i.e. they show a sharp increase in activity in the presence of lipid aggregates. . This activity increase has been correlated with the opening of an amphipathic helical lid-domain an amphiphilic α helix of about fifteen amino acids that covers the active site in the absence of substrate aggregates. [8-9]. the flexible lid-domain is attached to the core domain of the enzyme, which in most lipases features the α/β- hydrolase fold, [10-11]. Even though the sequence similarities between the various lipases is small. Among the bacterial lipases, when α helix covers the active site, the Enzyme is in its closed or inactive conformation. In this conformation, the hydrophobic face of the amphiphilic helix interacts with hydrophobic residues surrounding the active site while its hydrophilic face interacts with water molecules. The substrate cannot be interacting with the catalytic triad. In the open or active conformation of the enzyme which is a result of interfacial activation mechanism, there is a shift of α helix constituting the cover. The hydrophobic face of the helix facing inward before the active site exposed to the solvent, creating a hydrophobic surface, assumed to interact with the interface water / fat. The active site of the enzyme is then accessible to the substrate. The 46 kDa lipase from the animal skin pathogen Staphylococcus hyicus (SHL) is unique in having high A1 and minor A2 phospholipase activities, besides having considerable lipase activity. [15-16]. Based on the crystallographic data, some residues, different from the catalytic triad appear to be important in the catalytic mechanism. These residues form what is called the oxyanion hole. Their role is mainly to stabilize reaction intermediates, such as the tetrahedral intermediates. The hydrolysis of a carboxylic ester by the catalytic triad can be divided into six main stages. First, the carbon of the carboxylic function of the substrate undergoes nucleophile attack of the hydroxyl group of serine whose nucleophilicity is enhanced by the histidine residue following the formation of a hydrogen bond. The imidazole ring of histidine becomes protonated and positively charged. This positive charge is stabilized by a charge of an acidic residue (Asp or Glu) (Figure 1(1)). This results in the formation of the first tetrahedral intermediate, stabilized by two hydrogen bonds with residues of the oxyanion hole (Figure 1(2)). Subsequently, there is release of a molecule of alcohol, formation of the acyl-enzyme (Figure 1(3)) and nucleophile attack of the acyl-enzyme by a water molecule (Figure 1(4)). This second nucleophile attack results in the formation of a second tetrahedral intermediate, stabilized by the oxyanion hole (Figure 1(5)). Finally, there is the fatty acid release and return of the enzyme in its original conformation (Figure 1(6)) [12-14]. Hence, we have studied the use of Crocetine as a new treatment for Micrococcus hyicus with low secondary effects.
Materials and Methods:
Building the 3D structure of Crocetin:
The inhibitor model was built using Hyperchem software (www.hyper.com) based on Lewis structure. Atoms have been chosen from dialog box (default elements) in build menu. The 3D structure of the Crocetine is shown in Figure 2.
Optimizing the Structure of inhibitor model:
In this step, the inhibitor model structure was minimized by performing a molecular mechanics optimization using MM+ force field and Polack Ribier algorithm to obtain the most Stable structure geometry (Figure 1).
A single point calculation was performed in order to compute the energy and gradient of the inhibitor model, this method allowed the calculation of the total energy and gradient before the geometry optimization.
Enzyme structure optimization:
The crystal structure coordinates of the lipases enzyme (3HIH for Staphylococcus hyicus lipase and PDB_ID: 1HPL for pancreatic Horse lipase) was obtained from the Protein Data Bank (PDB) (http://www.rcsb.org). Water molecules, hetero atoms and ligands such as calcium and zinc ion were removed . All hydrogen atoms were added to the protein including those necessary to define the correct ionization and tautomeric states of amino acid residues using Hyperchem software. A two-step procedure was set up for the energy minimization of protein using the same software. In the first step, all hydrogen atoms in the protein were allowed to optimize. The hydrogen locations are not specified by the X-ray structure but these are necessary to improve the hydrogen bond geometries. In the second stage, all protein atoms were allowed to relax. Minimization in both stages was performed using 100 steps of steepest descent and 2000 Steps of conjugate gradient algorithm .
Docking procedure of Crocetin:
Docking of the inhibitor in the active site of the two lipases was carried out using GOLD 4.1.2 software (Genetic optimization of ligand docking). The procedure consisted of three main parts: (1) A scoring function to rank different binding modes; the Gold score function is a molecular mechanics-like function with four terms, was used. (2) A mechanism for placing the ligand in the binding site; GOLD uses a unique method to do this, which is based on fitting point. (3) A search algorithm to explore possible binding modes; GOLD uses a genetic algorithm (GA) [20-21]. This method allows a partial flexibility of protein and full flexibility of ligand  for each of the 10 independent GA runs.
Discussion and Results:
Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the lipase structure of the S. hyicus lipase at 2.86 Å resolutions. Staphylococcus hyicus lipase molecules has a heart-like shape with approximate dimensions of 60 Å-60 Å-50 Å, SHL has an α/β-hydrolase fold,9 with a central seven-stranded parallel β-sheet covered on one side by helices α1 and α14, and on the other side by helices α2, α4 and the short helix α13. As expected, Ser124, Asp314 and His355 form the catalytic triad. The edge of the active site is lined by helices α7, α12, and lid helices α8 and α9. The first two strands of the central β-sheet are absent compared to the canonical α/β-hydrolase topology.  Furthermore, the SHL topology also contains a b1-α3-b2 insertion between helix α4 and strand β5, the lipase is in an open conformation, with the active site partly covered by a neighboring molecule. Ser124, Asp314 and His355 form the catalytic triad, have been studied by consulting literature of previous works and also by using the software PyMOL to understand the specificity of the active site towards the substrate. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.
The results of docking have shown 10 binding possibilities of Crocetine in the active site of both lipases, and we have accepted one of them according to the weakest inter-atomic distance (ID) between the oxygen atoms of the hydroxyl group of the catalytic amino acid Ser (for 3HIH: Ser124; for 1HPL: Ser152) and the hydroxyl group of Crocetine. It is attempted that this distance allows the formation of covalent bond between these described atoms basing on the catalytic mechanism. In docking, it is admitted that the affinity of the inhibitor towards lipase is related reciprocally with the ID value (when the ID value decreases the affinity increases). For 3HIH, we have recorded an ID of 1.3 Å (Figure 3) which is 1 times smaller than the ID recorded for 1HPL, with a value of 1.4 Å (Figure 4). From these results, it is attempted that Crocetine presents a strong affinity to Staphylococcus hyicus lipase than to pancreatic Horse lipase.
Docking study of the natural inhibitor Crocetine Should Be Trying to take care for developing a New Treatment for Micrococcus hyicus, crocetin has presented a higher affinity towards Staphylococcus hyicus lipase than to pancreatic Horse lipase, based on ID values of both lipases. These results have shown the potential of crocetin as a new treatment with low side effects. Nonetheless, experimental in vitro and in vivo studies are required to demonstrate the effectiveness of the inhibitor. Therefore this study Reveal affinities of inhibitors to lipases which are considerably easier to implement, cheaper and faster compared to experimental methods.