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 hypothesis 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-binding sites. It was hypothesized that if any enzyme has similar or identical conserved domain in its catalytic region, the construct can bring similar catalytic activity. Using this approach, the region which covers such functional module has to be modeled for yielding enzyme constructs. The present approach has provided a high likelihood to design stable metalloenzyme constructs from the sequences of methanogens due to their low functional 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, enzyme 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 methanogens 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-  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 Cα (Ф) of paludies C5 (bootstrap value 987–1000) than with first amino acid residue and Cα to N (ψ) 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 sequence alignment (Shaded regions show highly variable amino acids and bolded regions represent functional amino 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) 1000 MethanosarcinamazeiGo1 (Supplementary). 875 MethanococcoidesburtoniiDSM6242 MethanosaetathermophilaPT Designing coenzyme F420 non-reducing hydroge- 987 Methanococcusmaripaludis nase. Archaeal genomes have consisted of 6 cobalt con- 997 MethanococcusmaripaludisS2 taining enzymes include coenzyme F420 hydrogenase, 998 MethanococcusmaripaludisC5(Model1) 1000 MethanococcusmaripaludisC6 methyl coenzyme M reductase, hydrogenase matura- MethanococcusvannieliiSB tion protease, carbon monoxide dehydrogenase, rubre- 792 Methanothermobacterthermautotrophicusstr.DeltaH doxin, urease, acetyl-CoA decarbonylase/synthase com- MethanobrevibactersmithiiATCC35061 442 MethanocorpusculumlabreanumZ(Model2) plex. Coenzyme F420 hydrogenase has two types of en-

zyme modules, such as coenzyme F420-reducing hydro- 0.05 genase and coenzyme F -non-reducing hydrogenase Fig. 2. Phylogenetic tree of SHCCC sequences obtained from metha- 420 nogens and closely related organisms (CFNRH). As similar to SHCCC, no crystallographic structures of these enzymes in the PDB are opted for en- formed second clade having organisms belonged to the zyme designing. The protein sequence of Methano- groups of halophilic and sulfur reducing archaea (Fig. sarcina acetivorans C2A under accession number NP_ 2). As the results of phylogenetic analysis, evolution- 616085 (construct) is suited on designing CFNRH con- based approach provides a high likelihood to design me- struct (Supplementary). As compared to coenzyme F420- talloenzymes. reducing hydrogenase and non-reducing hydrogena- Construct 2 is slightly better than construct 1 as it se, hydrogenase maturation protease is distantly rela- has the lowest binding energy (–9.79 kcal/mol). Both ted for its function. CFNRH of this study is phyloge- enzyme constructs are opted for catalytic conversion netically resembled to coenzyme F420-reducing hydro- uroporphyrin I and precorin-2-dihydro sirohydrochlo- genase of other methanogens (Fig. 3). It pointed out rin to the respective products (Table). The most im- that coenzyme F420-reducing hydrogenase (hysF) and portant interactions contributing to the high binding hydrogenase maturation protease (hycI) are distinctly affinity are six H-bonds between the carbonyl group of produced monophyletic clades in the phylogenetic sirohydrochlorin and the side chains of His09, Leu70, tree. There are separate sub-clusters for the species of Arg12 and Ser16 in construct 1. The possible interac- all genera including Methanococcus, Methanosaci- tion sites are: O2-His09 (3.10 C), O15-Leu70 (2.93 C), na, Methanospirillum and thermophilic archaea in

70 COBALT- AND NICKEL-CONTAINING ENZYME CONSTRUCTS FROM THE SEQUENCES OF METHANOGENS

Molecular docking for binding energy of SHCCC and CFNRH constructs-substrate complexes

Substrates Binding energy, kcal/mol Inhibition constant, mM Intermolecular energy, kcal/mol Internal energy, kcal/mol Sirohydrochlorin cobaltochelatase

Construct 1

Sirohydrochlorin –9.60 0.09* –10.08 –7.76 Siroheme –6.54 0.02 –10.44 –9.98

Uroporphyrin I –9.04 0.24* –9.96 –8.98

Uroporphyrin III –7.65 2.48* –8.55 –7.83 Coproporphyrin III –2.89 7.59 –5.97 –4.56

Precorin-2-dihydro –8.65 0.45* –8.83 –7.76 sirohydrochlorin Construct 2

Sirohydrochlorin –9.79 0.07* –10.09 –6.35

Siroheme –6.06 0.04 –9.89 –9.49

Uroporphyrin I –9.48 0.11* –10.19 –8.66 Uroporphyrin III –8.72 0.40* –9.68 –6.95

Coproporphyrin III –4.12 0.95 –7.06 –4.59

Precorin-2-dihydro –9.58 0.09* –9.94 –6.63 sirohydrochlorin

Coenzyme F420 non-reducing dehydrogenase Construct

Reduced coenzyme F420 –4.81 0.30 –10.11 –8.80 THF-L-glutamate –3.44 3.01 –8.64 –7.39

Reduced FMN –5.07 0.19 –4.80 –4.50

10-Formyltetrahydrofo- –4.31 0.70 –9.08 –7.03 lyl-L-glutamate

*Inhibition constant is expressed as µM. which many of the hydrogenases belonging to Metha- The single conserved domain (CbiX_SIR_B_N of nococcus genus. It may functionally diverge into NiFe- chelatase class II superfamily) is detected in SHCCC se- hydrogenase maturation protease of Candidatus Ko- quences of M. maripaludies and M. labreanum Z that rarchaeum cryptofilum OPF8 (Fig. 4). may catalyze biochemical reactions. A divergent evolu- Enzyme construct has shown strong interactions tion occurred among cobalt and nickel metabolisms with reduced FMN. It is also supported by computing due to more ancestral similarities of cobaltchelatase as inhibition constant (38.58 µmol) of reduced FMN as reported earlier [11]. Cobaltochelatase of archaeal do- shown in Table 1. Intermolecular energy of construct- main has shown more phylogenetic similarity with reduced coenzyme F420 complex is –12.26 kcal/mol. A CbiX of Bacillus megaterium [12]. Similarly, the phylo- strong binding affinity is observed in enzyme const- genetic analysis in this study revealed a phylogenetic ruct-reduced FMN wherein two H-bonds formed bet- resemblance between methanogens and B. megaterium ween the side chains of Lys80 and Ala86 and reduced for SHCCC sequences. It also closely related to halo- FMN. The possible interaction sites are: O1-Lys80 philic archaea. CbiX tolerates large sequence rearrange- (2.97 C) and O6-Ala86 (2.88 C) (Supplementary). ments in four out of the nine loop regions of the SHCCC.

71 CHELLAPANDI P., BALACHANDRAMOHAN J.

Fig. 3. Functional features of CNFRH enzyme constructs computed by multiple sequence alignment (Shaded regions show the highly variable amino acids and bolded regions represent functional amino acids)

These rearrangements may serve as starting points for terminal end of CbiX, the histidine rich region is likely the evolution of new functions (binding of new subst- to act as a cobalt store or could interact directly with the rates) [13]. It suggested that conservations of enzyme cobalt transport system to allow direct transfer of the function are not deviated at sequence level, and its ca- metal ion from the import system to its site of action. talytic function evolved from the structural features, Construct 1 has more numbers of histidine residues, particularly loop regions of these constructs. Thus, the some of which contributed for H-bonding with siro- present approach is more convenient for designing an hydrochlorin in accordance to the earlier report [17]. enzyme even from its sequence in appropriate manner. There is no common agreement on the catalytic me- Glu264 and His183 are mechanistically the most criti- chanism of Ni-Fe hydrogenase. The sequences of CFNRH cal residues for catalytic activity and form the active si- from M. acetivorans consisted of H2MP domain assig- te in conjunction with residues that selectively bind the ning function to hydrogenase specific C-terminal endo- tetrapyrrole substrate in accordance with the earlier re- peptidases. Phylogenetic analysis in this study also sup- ports [14, 15]. The results from molecular mechanics stu- ported its functional divergence with NiFe-hydrogena- dies implied that conformational free energies of our en- se maturation protease of Candidatus Korarchaeum cryp- zyme constructs are more stable. Hence, the structures of tofilum OPF8. H2MP domain may be originated from these constructs have to show low degree of freedoms to subsequent functional divergence during evolution. The recognize sirohydrochlorin specificity, which is rather maturation of FeNi-hydrogenases includes the forma- than other tetrapyrrole substrates (Supplementary). tion of nickel metallocenter, proteolytic processing and Removal of two protons followed by insertion of assembly with other subunits. The hydrogenase matu- the cobalt ion induces planarity into macrocycle, resul- ration endopeptidases are responsible for the proteolytic ting in its exclusion from the active site [16]. Thus, si- processing, liberating a short C-terminal peptide by clea- rohydrochlorin is bent such that two pyrrole nitrogens ving after His or Arg residue. This cleavage is nickel de- are orientated towards the active site histidine for its pendent. Similarly, in this study a strong binding affinity binding with SHCCC. Similarly, sirohydrochlorin stab- is observed between individual atoms of reduced FMN ly interacts with the side chains of His09, Leu70, and the side chains of enzyme construct. It reflects the Arg12 and Ser16 in construct 1 and Ser13 and Arg14 in en zyme mimics as similar to hydrogenase maturation construct 2. A strong interaction is found between con- endopeptidases by cleaving His87 or Arg84 residue in struct 1 and sirohydrochlorin in the side chain of Arg12. the C-terminal. Based on DFT studies, Pavlov et al. pro- At Ser13 and Arg14 residues, construct 2 forms a com- posed a mechanism stating the resting state of the di- plex with sirohydrochlorin. It indicates that either nuclear cluster is Ni (II) Fe (III); H2 first binds to Fe in Ser13 or Arg12 could serve as active region providing the form of a molecular hydrogen complex, which then catalytic function to these constructs. Towards the C- undergoes heterolytic splitting [18]. Hydride transfer

72 COBALT- AND NICKEL-CONTAINING ENZYME CONSTRUCTS FROM THE SEQUENCES OF METHANOGENS

772 HycI[Pyrococcushorikoshii OT3] 465 HycI[Pyrococcusfuriosus DSM3638] HycI[Thermofilumpendens Hrk5] 999 HysFdeltasubunit[Methanococcusmaripaludis C7] 913 HysFdeltasubunit[Methanococcusmaripaludis S2] 1000 HysFdeltasubunit[Methanococcusvannielii SB] 534 HysFdeltasubunit[Methanococcusaeolicus Nankai-3] HysFdeltasubunit[Methanocaldococcusjannaschii DSM2661] 912 HysFdeltasubunit[Methanosarcinabarkeri str.Fusaro] 517 325 996 HysFdeltasubunit[Methanosarcinabarkeri] 203 HysFdeltasubunit[Methanosarcinamazei Go1 282 HycI[CandidatusMethanoregulaboonei 6A8] 751 HysFdeltasubunit[Methanospirillumhungatei JF-1] 267 997 HycI[Methanospirillumhungatei JF-1] HysFdeltasubunit[Methanocorpusculumlabreanum Z] FrhD[Methanosphaerastadtmanae DSM3091] 346 386 999 HycI[Pyrococcusabyssi GE5] 629 FrdH[Pyrococcusabyssi GE5] 1000 HycI[Thermococcuskodakarensis KOD1] 613 749 FrdH[Methanosarcinaacetivorans C2A] Ni,Fe-hydrogenasematurationfactor[CandidatusKorarchaeumcryptofilum OPF8] 756 HycI[Methanospirillumhungatei JF-1] 807 HycI[Methanocorpusculumlabreanum Z] 941 HycI[Aciduliprofundumboonei T469] HycI[Methanocaldococcusjannaschii DSM2661] 823 HycI[Methanococcusaeolicus Nankai-3] 723 362 HycI[Methanococcusvoltae A3] HycI[Methanococcusvannielii SB] Fig. 4. Phylogene- HycI[Methanococcusmaripaludis S2] HycIpeptidase[Methanococcusmaripaludis C5] tic tree of CNFRH sequences obtai- 900 HycIpeptidase.Asparticpeptidase.MEROPSfamilyA31[Methanococcusmaripaludis C5] HycI[Methanococcusmaripaludis C6] ned from metha- 387 HycI[Methanococcusmaripaludis C7] nogens and closely related organisms 0.1 to Fe and proton transfer to the adjacent Cys thiolate Acknowledgements. The first author is thankful to the ligand is accompanied by decoordination of the proto- University Grants Commission, New Delhi, India, for fi- nated Cys thiol from Ni whi le remaining bound to Fe. nancial assistance (32-559/2006) to carry out the work. The cyanide ligand on Fe simultaneously binds with the П. Челла панді, Дж. Ба ла чан дра мо хан Ni atom in a bridging binding mode. After the H2 dis- sociation, the hydride bound to Fe can then be trans- Ко бальт- і нікель-вмісні фер ментні ко нструкції із ferred to Ni which should be a necessary preliminary послідов нос тей ме та но генів for subsequent H+ or electron transport. Accordingly, oxidation of molecular hydrogen could be mediated by Ре зю ме

CFNRH construct on reduced FMN. A strong phyloge- Мета. Ви ко ристати кон сер ва тивний до ме н, от ри маний із по- netic correspondence of these enzyme constructs only слідов нос тей ме та но генів, для ко нстру ю ван ня фер ментів. Особ- to other closely related methanogens implied its evolu- ливу ува гу приділено ме та ло фер мен там, оскільки вони ма ють важ- ливе ево люційне зна чен ня. Ме то ди. Теорія мо ле ку ляр ної ево люції, tionary conservation. ме то ди мо ле ку ляр но го мо де лю ван ня і мо ле ку ляр но го докінгу. Ре - Similar approach has already been reported for de- зуль та ти. Ко нстру ю ван ня ко бальт-вмісної ко баль то вої хе ла - та зи і нікель-вмісно го ко фер мен ту F не ре дукційної гідро ге на зи signing β-methylaspartate mutase [3] and formyltetra- 420 із кон сер ва тив них до менів, ото чу ю чих ме тал- і суб страт-зв’я зу - hydrofolate ligase [5] from the sequences of archaea вальні сай ти, здійсне но на основі теорії мо ле ку ляр ної ево люції. [19]. Similar to that, we have not done any alternation Зроб ле но при пу щен ня сто сов но того, що якщо будь-який фер - in native amino acids position or replacing residues, мент містить у своєму ка талітич но му сайті схо жий або іден - тич ний кон сер ва тив ний до мен, то ко нструкції може бути при- and, thus, it is directly based on the conserved domain та ман на подібна ка талітич на ак тивність. Ви ко рис то ву ю чи цей of metalloenzymes. підхід для ство рен ня фер мен тної ко нструкції, потрібно змо де -

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