Structural features of a cold-adapted Alaskan bacterial lipase

The three dimensional model of cold-adapted Alaskan psychrotroph Pseudomonas species (Strain B11-1) lipase has been constructed by homology modeling based on the crystal structure of acetyl esterase from Rhodococcus species and refined by molecular dynamics methods. Our model locates the substrate-binding cavity and further suggests that Ser-155, Asp-250, and His-280 are the members of the catalytic triad. Substrate specificity of the modeled lipase has been examined by docking experiments, which indicates that the ester of C(6) fatty acid has the highest affinity for the enzyme. Our model also identifies the oxyanion hole that plays an important role in the stabilization of the tetrahedral intermediate during catalysis. Comparison of this cold-adapted lipase with the crystal structure of a thermophilic Bacillus stearothermophilus P1 lipase supported the assumption that cold-adapted enzymes have a more flexible three-dimensional structure than their thermophilic counterparts. The conformational flexibility of this modeled cold-adapted lipase at low temperature probably originates from a combination of factors compared to its thermophilic counterpart, i.e., lower number of salt bridges and cation-pi interactions, increase in the non-polar surface area exposed to solvent. Our study may help in understanding the structural features of a cold-adapted lipase and can further be used in engineering lipase that can function at or near extreme temperatures with considerable biotechnological potential.     

Cloning and Expression of <Emphasis Type="Italic">lipP</Emphasis>, A Gene Encoding a Cold-Adapted Lipase from <Emphasis Type="Italic">Moritella</Emphasis> sp.2-5-10-1

A gene (lipP, 837 bp in length) coding for a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-5-10-1 isolated from Antarctic region was cloned and sequenced in this study. The deduced amino acid sequence revealed a protein of 278 amino acid residues with a molecular mass of 30,521. The primary structure of the lipase deduced from the nucleotide sequence showed consensus pentapeptide containing the active serine [Gly-Trp-Ser-Leu-Gly] and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. These sequences were involved in the lipase active site conformation. Structure factors that would allow proper enzyme flexibility at low temperatures were discussed. It was suggested that the changes in the primary structure of the psychrophilic lipases compared to the thermophilic ones could account for their ability to catalyze lipolysis at temperatures close to 0°C. For expression, the sequence corresponding to the cold-adapted lipase of strain 2-5-10-1 was subcloned into the pET-28a expression vector to construct a recombinant lipase protein. Expression of the lipase by Escherichia coli BL21 (DE3) cells was observed as clear halos on 1% (vol/vol) tributyrin upon induction with IPTG at 25°C.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Cloning of triose phosphate isomerase gene from an antarctic psychrophilic <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. by degenerate and splinkerette PCR

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.     

Cyanobacterial Photosystem II at 3.2 Å resolution – the plastoquinone binding pockets

Photosystem II from thylakoid membranes of the thermophilic cyanobacterium Thermosynechococcus elongatus was solubilized with n-β-dodecylmaltoside and purified using anion exchange chromatography. Molecular weight, pigment stoichiometry and subunit composition were assayed using various techniques. The holocomplex is dimeric with a molecular mass of 756 ± 18 kDa and functionally fully active. Crystals obtained from these samples showed significantly improved quality leading to a 3D structure at 3.2 Å resolution. Several loop regions of the principal protein subunits are now defined that were not interpretable at lower (3.8 Å) resolution, thus resulting in a more complete model. The head groups of the cofactors of the electron transfer chain and of the antennae have been modeled, coordinating and hydrogen bonding amino acids identified and the nature of the binding pockets derived. The orientations of these cofactors resemble those of the reaction centre from anoxygenic purple bacteria. For the two plastoquinones, electron density was only found for the head group of Q_A and none for Q_B indicating low or even no occupancy of this site in the crystal structure. Both binding pockets and problems related to the Q_B site are discussed here and compared to the situation in the purple bacterial reaction centre.     

Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability

The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k _cat /K _m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t _1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T _m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.     

Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm

A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine -class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.Figure: A three-dimensional (3D) structure of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. This modeled 3D structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668).     

<Emphasis Type="Italic">Fervidobacterium changbaicum</Emphasis> Lip1: identification,cloning, and characterization of the thermophilic lipase as a new member of bacterial lipase family V

A novel lipase gene encoded 315 amino acid residues was obtained using lipase-prospecting primers and genome walking from hyperthermophilic bacterium Fervidobacterium changbaicum CBS-1. Sequence alignment and phylogenetic analysis revealed this novel lipase is a new member of bacterial lipase family V. The recombinant enzyme F. changbaicum lipase 1 (FCLip1) showed maximum activity at 78°C and pH 7.8. It displayed extreme thermostability at 70°C and was also stable across a wide pH range from 6.0 to 12.0. Kinetic study demonstrated FCLip1 preferentially hydrolyzed middle-length acyl chains, especially p-nitrophenyl caprate and tricaprylin. With p-nitrophenyl caprate as a substrate, the enzyme exhibited a K _m and k _cat of 4.67 μM and 22.7/s, respectively. In addition, FCLip1 was resistant to various detergents and organic solvents. This enzyme is the first reported thermophilic lipase from bacterial family Thermotogaceae. Its extreme stability with respect to temperature and pH, along with its triglyceride hydrolysis activity, indicate that FCLip1 has high potential for future application.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Electric field control of lipase membrane activity

Lipase (EC 3.1.1.3., from Pseudomonas sp.) was entrapped in collagen membrane containing liquid crystal (4-methoxybenzilidene-4′-n-butylaniline). The activity of the lipase–liquid crystal membrane at an applied voltage of 4 V was 3.4 compared to a membrane tested without imposition of an external electric field. A linear relationship was observed between the activity of the lipase–liquid crystal membrane and the current. The apparent Michaelis constant (K′_m) of the lipase–liquid crystal membrane under electric field was identical to that of the membrane under ordinary condition. Activation of the lipase–liquid crystal membrane was observed repeatedly, i.e., activation in the presence of an electric field and reversion to a basal level upon removal of the field occurred cyclically. Activity control of immobilized enzymes is desirable for switching devices of a bioreactor. Possible mechanisms of the lipase activation by electric field are discussed.     

Improved Model of a LexA Repressor Dimer Bound to recA Operator

Abstract

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Δ_1–67 S119A, S119A, G85D and Δ_1-67 quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the ‘cleavable’ cleavage site region found in the crystal structures is actually the more relevant form of the region in wildtype LexA since it agrees with the value of the pre-exponential Arrhenius factor for its auto- cleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a ‘conformational switch’ inferred from the crystal structures is meaningless.     

Substrate Specificity and Mode of Action of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Substrate specificity of the lipase of thermophilic fungus, Humicola lanuginosa S–38, was investigated. It was found that the lipolytic activity was greatly influenced by the structure of both fatty acid and alcohol moieties of the substrate. It was concluded that the hydrolysis of both water soluble and water insoluble ester was catalyzed by the Humicola lipase itself. The Humicola lipase showed no positional specificity and split ester bonds on all positions of triolein at about the same rate. Both palmitic acid (α) and linoleic acid (β) ester bonds of phosphatidyl-ethanolamine were split indicating no positional specificity of fatty acid ester bonds. From above results, it was made clear that mode of action of Humicola lipase on triolein and on phosphatidyl-ethanolamine is identical. The Humicola lipase had no activity of lipoprotein lipase.     

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. III. Four cold-adapted,peridinin-containing taxa and the presence of trigalactosyldiacylglycerol as an additional glycolipid

Despite their importance in marine and freshwater microalgal assemblages, cold-adapted dinoflagellates have been the subject of few comprehensive lipid studies, particularly with respect to those lipids that comprise plastid membranes. In an effort to understand the differences between warm- and cold-adapted dinoflagellate glycolipid composition, four peridinin-containing, cold-adapted dinoflagellates were surveyed for intact forms of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two common plastid lipids, using positive-ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). It was determined that the dominant forms of MGDG and DGDG in these cold-adapted, peridinin-containing dinoflagellates possessed C₁₈ fatty acids and did not, with the exception of a 20:5/18:5 form of DGDG in a cold-adapted Gymnodinium sp. from the Baltic Sea, have C₂₀ fatty acids. This finding is in contrast to an earlier study of 35 peridinin-containing, warm-adapted dinoflagellates, which discovered a cluster dominated by C₁₈ fatty acids and a cluster dominated by both C₂₀ and C₁₈ fatty acids. The key difference in MGDG and DGDG production between the former group and the cold-adapted dinoflagellates examined in this study is that the cold-adapted species’ DGDG fatty acids were less saturated. Each cold-adapted dinoflagellate possessed both 18:5/18:5 and 18:5/18:4 DGDG, while most of the warm-adapted dinoflagellates contained only 18:5/18:4 DGDG. This survey also revealed the presence of a putative 18:1/14:0 trigalactosyldiacylglycerol (TGDG) as a dominant glycolipid in Gymnodinium sp. TGDG, previously unreported in dinoflagellates, was also discovered in Gymnodinium sp. in the forms of 18:1/16:0 and 18:1/18:1 TGDG, as minor lipids. Since the fatty acids associated with TGDG are not those found with dominant forms of MGDG or DGDG, TGDG may be produced by a different biosynthetic pathway.     

(S)-1-(Pent-4′-enoyl)-4-(hydroxymethyl)-azetidin-2-one derivatives as inhibitors of human fatty acid amide hydrolase (hFAAH): synthesis,biological evaluation and molecular modelling

Abstract

A series of lipophilic ester derivatives (2a–g) of (S)-1-(pent-4′-enoyl)-4-(hydroxymethyl)-azetidin-2-one has been synthesised in three steps from (S)-4-(benzyloxycarbonyl)-azetidin-2-one and evaluated as novel, reversible, β-lactamic inhibitors of endocannabinoid-degrading enzymes (human fatty acid amide hydrolase (hFAAH) and monoacylglycerol lipase (hMAGL)). The compounds showed IC₅₀ values in the micromolar range and selectivity for hFAAH versus hMAGL. The unexpected 1000-fold decrease in activity of 2a comparatively to the known regioisomeric structure 1a (i.e. lipophilic chains placed on N₁ and C₃ positions of the β-lactam core) could be explained on the basis of docking studies into a revisited model of hFAAH active site, considering one or two water molecules in interaction with the catalytic triad.     

Isolation and Identification of the Lipolytic and Thermophilic Fungus

In view point of the basic research for the enzyme properties and the development of utilization of lipase, a thermophilic fungus which could produce a remarkable amount of thermostable, alkalistable and extracellular lipase has been isolated from the compost soil. The taxonomical characteristics of this thermophilic fungus were examined and it was identified as Humicola lanuginosa S-38.     

马蜂肠道菌的分离鉴定及产消化酶功能菌株的筛选

【背景】马蜂(Vespa mandarinia Smith)可以防治多种田间害虫,还具有药用价值,其肠道菌群结构和功能还有待研究。【目的】获得马蜂肠道可培养细菌并筛选出具有产消化酶功能的菌株,为理解肠道菌对宿主的影响机理及功能菌株的利用提供科学依据和研究材料。【方法】采用传统细菌分离培养法获得马蜂肠道菌,结合形态学以及16S rRNA基因序列分析进行鉴定;利用水解圈法分别筛选产蛋白酶、脂肪酶、淀粉酶和纤维素酶菌株;通过测量水解圈D与菌落直径d的比值,比较不同细菌的产酶能力。【结果】在马蜂肠道中共分离出6属10种细菌,其中芽孢杆菌属5种,肠球菌属、葡萄球菌属、明串珠菌属、乳球菌属和不动杆菌属各1种。从获得的61个菌株中筛选出6个具有产消化酶功能的菌株。其中,苏云金芽孢杆菌V44具有产蛋白酶、淀粉酶、脂肪酶和纤维素酶4种消化酶的能力;粪肠球菌V6具有产淀粉酶、蛋白酶和脂肪酶3种消化酶的能力;蜡样芽孢杆菌V43具有产蛋白酶、淀粉酶和纤维素酶3种消化酶的能力;粪肠球菌V20、蜡样芽孢杆菌V19和维德曼氏芽孢杆菌V22均具有产蛋白酶的能力。【结论】马蜂肠道细菌资源较丰富,部分有产消化酶的功能,可帮助马蜂消化食物,对宿主健康有一定影响。本研究筛选的6个菌株都能产蛋白酶,其中菌株V43和V44分别具有最强产淀粉酶和脂肪酶的能力,是可进一步开发利用的肠道功能菌株资源。     

In silico and empirical approaches toward understanding the structural adaptation of the alkaline-stable lipase KV1 from Acinetobacter haemolyticus

Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.     

Production and properties of an alkaline,thermophilic lipase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> NS2W

Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production. Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design. Cell growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium. Maximum lipase yields were 69.7 and 68.7 U ml⁻¹, respectively. The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained in the basal medium. The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3–11 with more than 70% activity retention. The lipase had an optimal activity at 55°C and was stable up to 60°C with more than 70% activity retention for at least 2 h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 344–348 DOI: 10.1038/sj/jim/7000254 Received 06 September 2001/ Accepted in revised form 15 March 2002     

Structure and function of the putative thioredoxin 1 from the thermophilic eubacterium Thermosipho africanus strain TCF52B

The thioredoxin system is a ubiquitous oxidoreductase system that consists of the enzyme thioredoxin reductase (TrxR), its cofactor nicotinamide adenine dinucleotide phosphate (NAD(P)H) and the protein thioredoxin (Trx). The system has been comprehensively studied from many organisms, such as Escherichia coli (E. coli); however, structural and functional analysis of this system from thermophilic bacteria has not been as extensive. In this study, Thermosipho africanus, a thermophilic eubacterium, Trx1 (TaTrx1) was successfully cloned, overexpressed and purified, to greater than 95% purity. Inspection of the amino acid sequence of TaTrx1 categorized the protein as a putative Trx. Its ability to reduce the interchain disulfides of insulin, in the presence of dithiothreitol, provided further evidence to suggest that it was a Trx. The three dimensional structure of the protein, determined using X-ray crystallography, provided additional evidence for this. The crystal structure was solved in space group P2₁2₁2₁ to 1.8 Ă resolution and showed the characteristic thioredoxin fold; four β-strands surrounded by three α-helices. The active site of TaTrx1 contained two cysteines that formed a disulfide bridge, and was structurally similar to the active site of EcTrx1. Further studies indicated that TaTrx1 was far more stable than Trx1 of E. coli (EcTrx1). The protein could withstand both higher temperatures and higher concentrations of guanidine hydrochloride before denaturing. Our studies have therefore identified a novel thermophilic putative Trx that structurally and functionally behaves like a Trx.     

Homology modeling of human histone deacetylase 10 and design of potential selective inhibitors

Abstract

Histone deacetylases (HDACs) are implicated in the pathology of various cancers, and their pharmacological blockade has proven to be promising in reversing the malignant phenotypes. However, lack of crystal structures of some of the human HDAC isoforms (e.g., HDAC10) hinders the design of the isoform-selective inhibitor. Here, the recently solved X-ray crystal structure of Danio rerio (zebrafish) HDAC10 (Protein Data Bank (PDB) ID; 5TD7, released on 24 May 2017) was retrieved from the PDB and used as a template structure to model the three-dimensional structure of human HDAC10. The overall quality of the best model (M0017) was assessed by computing its z-score—a measure of the deviation of the total energy of the structure with respect to an energy distribution derived from random conformations and by docking of known HDAC10 inhibitors to its catalytic cavity. Furthermore, to identify potential HDAC10-selective inhibitor ligand-based virtual screening was carried out against the ZINC database. The free modeled structure of HDAC10 and its complexes with quisinostat and the highest-ranked compound ZINC19749069 were submitted to molecular dynamics simulation. The comparative analysis of root-mean-squared deviation, root-mean-squared fluctuation, radius of gyration (Rg), and potential energy of these systems showed that HDAC10-ZINC19749069 complex remained the most stable over time. Thus, M0017 could be potentially used for structure-based inhibitor against HDAC10, and ZINC19749069 may provide a scaffold for further optimization.

Communicated by Ramaswamy H. Sarma