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Cobalt- and Nickel-Containing Enzyme Constructs from the Sequences of Methanogens

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Cobalt- and Nickel-Containing Enzyme Constructs from the Sequences of Methanogens ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 1. P. 68–74 BIOINFORMATICS UDC 577.151.7 Cobalt- and Nickel-containing enzyme constructs from the sequences of methanogens P. Chellapandi, J. Balachandramohan Department of Bioinformatics, School of Life Sciences, Bharathidasan University Tiruchirappalli-620024, Tamil Nadu, India [email protected] Aim. The conserved domain of sequences revealed in methanogens is considered for designing enzymes among which the attention has been focused on the metalloenzymes showing evolutionary significances. Methods. Mo- lecular evolution, molecular modelling and molecular docking methods. Results. Molecular evolutionary hypo- thesis has been applied for designing cobalt-containing sirohydrocholine cobalt chelatase and nickel-contai- ning coenzyme F 420 non-reducing hydrogenase from conserved domains encompassing metal- and substrate-bin- ding sites. It was hypothesized that if any enzyme has similar or identical conserved domain in its catalytic re- gion, the construct can bring similar catalytic activity. Using this approach, the region which covers such func- tional module has to be modeled for yielding enzyme constructs. The present approach has provided a high li- kelihood to design stable metalloenzyme constructs from the sequences of methanogens due to their low functio- nal divergence. The resulted enzyme constructs have shown diverse reaction specificity and binding affinity with respective substrates. Conclusions. It seems to provide a new knowledge on understanding the catalytic compe- tence as well as substrate-specificity of enzyme constructs. The resulted enzyme constructs could be experimen- tally reliable as the sequences originally driven from methanogenic archaea. Keywords: molecular docking, metalloenzymes, conserved domains, molecular evolution, enzyme design, enzy- me constructs. Introduction. Enzymatic or microbial transformations ning chemical catalysts. The first step towards desig- are environmental friendly, clean process and useful ning a working catalyst is, therefore, knowledge on the means for obtaining biologically important compo- structure of the enzyme and its active site [2, 3]. Evolu- unds. Enzymes from nature rarely have the combined tionary conservation in sequence and structure would properties necessary for the industrial fine chemical make contribution in enzyme catalysis. Such conserved production that will be present over the course of a ma- amino acid residues are major concern in designing me- nufacturing process. Enzymes from extremophilic or- talloenzymes. ganisms may provide useful biocatalysts and may be Upon designing enzyme constructs, the molecular evo- even more valuable for biotransformation reactions. A lution of enzymes is playing a crucial role in biological range of methanogenic archaeon enzymes application systems when enzymes are in action [4, 5]. Thus, the pre- and usage of the organisms themselves in biotechnolo- sent study describes the application of a molecular evolu- gy are more restricted. At present, many studies explo- tionary hypothesis to design metalloenzymes constructs re the identification of novel enzymes from methano- from conserved domains in the sequences of methano- gens for the industrial application [1, 2]. gens encompassing metal- and substrate-binding sites. Metals are tightly bound in the active sites of me- Materials and methods. Evolutionary conserva- talloenzymes to bring the chemical activity. The metal- tion analysis of metalloenzymes. Complete protein se- loenzymes are potentially very good models for desig- quences of archaeal cobalt and nickel-containing en- Ó Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012 zymes were retrieved from GenPept of NCBI. Conser- 68 COBALT- AND NICKEL-CONTAINING ENZYME CONSTRUCTS FROM THE SEQUENCES OF METHANOGENS ved domains architecture of these sequences was sear- Molecular docking of enzyme construct-substrate ched in NCBI-Conserved Domain Database (CDD). complex. Substrate structures were retrieved from KEGG The metal binding sites of the sequences were iden- database with SIMCOM tool (http://www.genome.jp/ tified from availed PDB structures using the corres- tools/simcomp) and then molecular formats prepared ponding PSSM ID in NCBI-CDD. A position-specific as PDB format for molecular docking studies. Homolo- score matrix prepared from the underlying conserved gy models resulted from Prime program was prepared domain alignment was compared with the query sequ- with charges for molecular docking studies. Both sub- ences. Multiple sequence alignment of the selected se- strate and protein were treated with AMBER force field quences was performed by ClustalX 2.0 software [6], implemented in AutoDock 4.0. software. Ligand Fit do- in which aligned sequences were manually inspected to cking was conducted by AutoDock software with Ge- delete the low scoring sequences. After that, the neigh- netic Algorithm and computed inhibition constant, bin- bor joining phylogenetic tree was constructed by ME- ding energy and other molecular forces of resulted do- GA 4.0 software [7] with 1000 bootstraps values. cking models. Active site residues in enzyme const- Molecular modelling and enzyme designing. Homo- ruct as in energy grid were allowed to interact with sub- logy modelling was carried out by ModWeb, an auto- strate. After docking, enzyme-substrate complex was matic comparative protein modelling server, from que- solvated with explicit periodic boundary solvation mo- ry sequences obtained from methanogens [8]. A suitab- del. The quality of each docking model was checked by le PDB template for homology modelling was identifi- AutoDock scoring system and graphically represented ed with PSI-BLAST tool [9] by searching against PDB by PyMOL software. database. ProFunc server [10] was used to predict the Results and discussion. Designing sirohydrocholi corresponding function sites of models. The catalytic ne cobalt chelatase. Cobaltochelatase, methionine syn- domains of every protein model were compared with the thase, methionine aminopeptidase, methylmalonyl-CoA crystallographic protein structures. Amino acid residu- mutase and methyl aspartate mutase are cobalt-dependent es encompassing the sites for metal-binding and subst- enzymes identified in the genomes of archaea. Among rate-binding regions were removed from atomic coordi- these, sirohydrocholine cobaltochelatase (SHCCC) and nates of a protein model and subjected to further homo- anaerobic chelatase (ACC) are present in methanogens. logy modelling. Prime program in Maestro software The limited structural information is available for co- package (Schrodinger Inc.) was used to build the homo- balt chelatase in the PDB to date. The sequence identity logy models from the above selected regions and then of SHCCC is ranged from 30–46 % with PDB templa- evaluated with Structural Analysis and Verification Ser- tes. Enzyme sequences under accession numbers YP_ ver (SAVS) (http://nihserver.mbi.ucla.edu/SAVES/). 001098022 (construct 1) from Methanococcus maripa- The best scoring models were superimposed on the cor- ludies C5 and YP_001030522 (construct 2) from Me- responding PDB template by DALITE server (http:// thanocorpusculum labreanum Z have significant model- ekhidna.biocenter.helsinki.fi/dali_lite/start). Standard ling scores, and encompassed the shortest metal bin- dynamics simulation cascade module in Discovery Stu- ding sites incorporating functional region (Supplemen- dio software with CHARMM force field, steepest des- tary). Aligned SHCCC sequences have also more con- cent and adopted basis Newton-Raphson algorithms servation at the substrate- and metal-binding regions was used for generating structural conformers of each (Fig. 1). Construct 1 is evolutionarily related only to model. Conformers were typically created for mole- closely related species of the same genera and its func- cules that have a small number of alternate locations for tion may conserve in the particular sequence position a small subset of the atoms. Distance constraint of each and/or domain. SHCCC in M. labreanum Z shows mo- model was fixed between N-terminal to C-terminal, re evolutionary relationship with SHCCC of M. mari- and dihedral restraint was started from C to Ca (Ф) of paludies C5 (bootstrap value 987–1000) than with first amino acid residue and Ca to N (y) of second ami- other archaea. The different species of Methanosarcina no acid residue until the last amino acid residue in a mo- genus are clustered with the members in the genus of lecular dynamic ensemble. Methanococcus in the first clade, and then both of them 69 CHELLAPANDI P., BALACHANDRAMOHAN J. Fig. 1. Functional features of SHCCC enzyme constructs computed by multiple sequ- ence alignment (Shaded re- gions show highly variable amino acids and bolded regi- ons represent functional ami- no acids) 984 Sulfolobussolfataricus SulfolobusacidocaldariusDSM639 O7-Ser16 (2.96 C), O11-Arg12 (3.19 C), O5-Arg12 186 1000 HalobacteriumsalinarumR1 (2.78 C), and O5-Arg12 (3.38 C). Among these, Arg12 1000 HaloarculamarismortuiATCC43049 forms three H-bonds to confer higher binding affinity. Bacillusmegaterium 378 HaloarculamarismortuiATCC43049 Six H-bonds are formed between sirohydrochlorin and 109 1000 Methanosarcinaacetivorans construct 2 at atomic positions of H3-Ser13 (2.52 C), 1000 MethanosarcinaacetivoransC2A O6-Ser13 (3.08 C), O1-Arg14 (3.00 C), O5-Arg14 Methanosarcinabarkeristr.Fusaro 999 377 Methanosarcinamazei (2.98 C), O12-Lys19 (2.85 C), and O16-Lys19 (2.76 C)
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