Article

BIOINFOMATICS

DOI: 10.5504/bbeq.2011.0155

IN SILICO STRUCTURAL DETERMINATION OF GPAT ENZYME FROM OSTREOCOCCUS LUCIMARINUS FOR BIOTECHNOLOGICAL APPLICATION OF MICROALGAL BIOFUEL PRODUCTION
Maitree Baral, Namrata Misra, Prasanna Kumar Panda and Manakkannan Thirunavoukkarasu Council of Scientific and Industrial Research, Institute of Minerals and Materials Technology (formerly Regional Research Lab), Bioresources Engineering Department, Bhubaneswar, Orissa, India Correspondence to: Prasanna Kumar Panda E-mail: pkpanda@immt.res.in, pkrpan@gmail.com

Glycerol-3-phosphate acyltransferase (GPAT) is an enzyme in the triacylglycerol (TAG) biosynthetic pathway that catalyses the conversion of glycerol-3-phosphate to lysophosphatidic acid. Targeting key enzymes involved in TAG pathway is considered to be a powerful strategy for augmented lipid accumulation in microorganisms. In the present study three-dimensional structure of the marine microalgae, Ostreococcus lucimarinus GPAT protein was developed based on the crystal structure of Cucurbita moschata GPAT protein. Besides, several structure validation tools were employed to confirm the reliability of the developed model. The predicted and validated model reveals the tertiary structure of GPAT monomer comprising of two domains, the smaller domain I, which folds into a four helix bundle, and the larger domain II, which is constructed from alternating / secondary structural elements that give rise to 9-stranded sheet flanked by 11 helices. Critical structural analysis of the developed model reveals the presence of H(X) 4D motif; the latter being, a consensus sequence conserved amongst many glycerolipid acyltransferase. The detected cluster of positively charged residues H189, K243, H244, R285 and R287 in the model could be conjectured to be important in glycerol-3-phosphate recognition. The structural insight obtained from this in silico study may provide useful clues to further advanced biotechnological studies of strategic site-specific genetic and metabolic engineering of microalgae for enhanced biofuel production. Biotechnol. & Biotechnol. Eq. 2012, 26(1), 2794-2800 Keywords: homology modeling, molecular dynamics, GPAT, Ostreococcus lucimarinus, microalgae, biofuel, TAG pathway could lead to development of microalgal strains rich in oil content (6, 26, 30).

ABSTRACT

Introduction

The present global inconsistency in price as well as supply of fossil fuel based petroleum products and the consequent environmental damage caused by green-house gas emissions have triggered widespread interest amongst researchers to search for biomass-based renewable sources of energy (28, 38). In recent years liquid biofuel from microalgae has been demonstrated to be one of the eminently suitable feedstocks for production of biodiesel, although the process is too far away from being carbon neutral or commercially competitive (29, 32). Several laboratory and pilot-scale studies of algal biofuel production using raceway ponds as well as controlled photobioreactor systems have conclusively indicated the need for improvements in microalgal oil production through various approaches viz., biochemical, genetic and transcription factor engineering (6, 11). Biochemical engineering strategy involves applying artificial physiological stress such as nutrient depletion to channel metabolic flux to enhance lipid biosynthesis. However, the major limitation of this approach is the retarded cell growth and lower yield of biomass. With the recent advancements in the area of genomics and biotechnology, identification and over expression or knockout of candidate genes through genetic engineering strategies 2794

Fig. 1. Triacylglycerol biosynthesis pathway in microalgae.

Three major steps of triacylglycerol (TAG) biosynthesis involve sequential transfer of acyl group from acyl CoA to a glycerol backbone by glycerol-3phosphate acyltransferase (GPAT), lysophosphatidic acid acyl transferase (LPAT), phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT) respectively (4, 18). Glycerol-3phosphate (1)-acyltransferase initiates the TAG biosynthesis process with the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl CoAs into the sn-1 position of glycerol 3- phosphate to yield 1-acylglycerol3-phosphate/lysophosphatidic acid (LPA) (36, 37). The LPA is then condensed, catalysed by LPAT, with another acyl-CoA to produce phosphatidate (PA) which is further dephosphorylated by PAP to produce diacylglycerol. Finally, DGAT incorporates the third acyl-CoA into the diacylglycerol molecule to produce TAG (Fig. 1). Biotechnol. & Biotechnol. Eq. 26/2012/1

Previous reports indicate that genes involved in TAG assembly play an important role in total seed oil production (29). This is further corroborated by several experimental reports in which over expression of GPAT, LPAT or DGAT in oil yielding plants resulted in increased lipid accumulation (19, 20, 23, 34, 37, 40, 41). Together these studies suggest that engineering the above enzymes could accelerate the process of production of lipid in microalgae. Therefore in the present study, as a fundamental step in this direction, three dimensional structure of GPAT protein from Ostreococcus lucimarinus has been determined employing homology modeling. To our knowledge this is the first attempt to evaluate the structural details and provide insight into the functional key residues of GPAT from microalgae. The developed model would be useful as a reference structure of an enzyme of such a class until an experimentally determined structure is available.

integration time step of 2fs. The cut-off for the non-bonding interactions was 10 and the long range electrostatics were treated with the Particle mesh Ewald method. Finally, the graph was plotted by taking the root mean square deviation (RMSD) of structures generated during minimization and equilibration methods on Y-axis with time in ps on X- axis. Structural assessment of the developed model The reliability of the stable 3-D model was evaluated using various structure validation tools viz., PROCHECK (24), WHATCHECK (17), Verify 3D (9), ERRAT (5) and PSQS (21). Structural and packing architecture of the modeled structure were computed by calculating the total volume, accessible surface area, backbone and side chain dihedral angle, hydrogen bonding partners and hydrogen bond energy using VADAR 1.4 programme (39). The developed tertiary structure of GPAT was submitted to Protein Model Data Base (PMDB) (3); the latter being a public repository of computationally determined 3-D models of protein. Secondary topology prediction and active site identification STRIDE (10) which uses hydrogen bond energy and main chain dihedral angles to recognize helix, coils and strands, was subsequently employed to predict the secondary structure composition of the modeled GPAT protein. Identification of substrate accessible pockets and functional residues present in the active sites of the protein were determined with Computed Atlas of Surface Topography of Proteins (CASTp) programme (8).

Materials and Methods

Homology model building The tertiary structure of O. lucimarinus GPAT protein was generated using MODELLER 9v9 (31). A BlastP (1) search was performed against the PDB database (2) to find templates with maximum identity and lower E-value for the target sequence. Finally the crystal structure coordinates of Cucurbita moschata (Squash) GPAT at 1.90 (PDB ID: 1K30: chain A) and having 46% sequence identity was used as structural template for modeling. The multiple sequence alignment highlighting the conserved regions of the target and template along with other GPAT homologues were generated using the ClustalX (35) programme of BioEdit (13). Illustrations of the 3-D structures were viewed and edited by protein visualisation and manipulation tools like Swiss-PdbViewer (12) and UCSF Chimera (27). Molecular simulation and dynamics GROMACS 4.5 (http://www.gromacs.org) package (15, 25) was used for molecular simulation and dynamics. The protein molecule was solvated in the TIP3P water box (22) with each side measuring 10 from the edge of the complex. Na+ ions as counter ions were added to neutralise the charge of the system. The Particle Mesh Ewald method (7) was used for computing long range electrostatic interactions. The Linear Constraint Algorithm (16) was applied to fix all bond lengths in the system. The solvated system was energy minimised prior to the molecular dynamics (MD) simulation. In each of the steps energy minimization was executed by the steepest descent method for the first 1000 steps and then conjugated gradient method for the subsequent 1000 steps. To avoid unnecessary distortion of the protein, the system was subjected to an equilibration run of 50-100 ps and temperature and pressure controlled with the Berendsen weak coupling algorithm, during which all heavy protein atoms are restrained to their starting positions while the water is relaxing around the structure. Subsequently, the minimised structure was subjected to a 1000 ps MD simulation at 300K and 1 atmospheric pressure with an Biotechnol. & Biotechnol. Eq. 26/2012/1

Fig. 2. Sequence alignment of template (1K30_A) and target (GPAT).

Results and Discussion

Model building and Molecular dynamics The plethora of biological data brought about by the complete genome sequencing projects has fostered an unprecedented level of new biomedical, environmental and biotechnological applications. Proteins are responsible for mediating functional mechanisms of an organism, and all these vital functions are mainly determined by the 3-D structure of proteins. 2795

Fig. 3. Multiple sequence alignment of selected GPAT homologues as predicted from BlastP search (C. moschata (Cucurbita moschata), O. lucimarinus (Ostreococcus lucimarinus), J. curcas (Jatropha curcas), A. thaliana (Arabidopsis thaliana), O. sativa (Oryza sativa), Z. mays (Zea mays), C. reinhardtii (Chlamydomonas reinhardtii) and O. tauri (Ostreococcus tauri)). The H(X)4D motif found conserved in all GPAT protein sequences (square box); the residues determined to play a crucial role in the binding of glycerol-3-phosphate moiety corresponding to O. lucimarinus GPAT H189, K243, H244, R285, R287, E192 and D194 (solid black arrows); identical residues (asterisk); conserved changes (colon); weakly conservative changes (dot).

Notwithstanding the advancements in the areas of analytical techniques, determination of protein tertiary structure through X-ray crystallography and NMR spectroscopy is still one of the most time consuming efforts in biology. Consecutively the gap between the availability of sequence information and protein structure is exponentially increasing. However, with the development of various bioinformatics tools and programmes, 2796

in silico protein models from its amino acid sequence are considered to be at par in accuracy with experimentally determined structures. The homology model of GPAT was obtained through MODELLER 9v9 server using 1.90 resolution structure of GPAT protein from Cucurbita moschata (PDB ID: 1K30: Biotechnol. & Biotechnol. Eq. 26/2012/1

chain A) as template. Fig. 2 shows the conserved regions of the developed model and template as determined by pair wise sequence alignment and Fig. 3 reveals the pattern of sequence conservation among GPAt protein sequences from eight different organisms. Molecular simulation and dynamics is a very powerful toolbox in molecular modeling that optimizes structure by lowering the energy of biological macromolecules. Lower energy signifies thermodynamically stable structure. The RMSD and energy of the minimized protein structure were calculated and shown in Fig. 4 and Fig. 5 respectively. Results of the MD show that during the first 100-400 ps, the RMSD values have sizeable variations; thereafter, they reach equilibrium and remain stable. in addition total energy of the system over the course of the MD simulations indicates that all the simulated systems are stable.

amino acids) shows similar topology and active site structure with the crystal structure of the template (Fig. 6). it consists of 14 helices and 9 strands, which account for 43% and 11% of the total polypeptide chain respectively. in addition there are five segments of 310 helix (Residues: M14-S17, F159-R161, l225-A227, K242-h244, and Y329-i331). the secondary structural elements of GPAt protein are organised into two compact domains (36). in the modeled protein domain i, the smaller of the two, is constructed from the first 84N terminal residues that are folded to form a four helix bundle [1 (S21-R34), 2 (P43-D62), 3 (E67-t84) and a 310helix (M14-S17)]. A long loop region links domain i to domain ii, the later being constructed from alternating / secondary structural elements that give rise to a 9 stranded mixed parallel/anti-parallel sheet [1 (S2134-h136), 2 (I163-R165), 3 (N182-G187), 4 (I214-A217), 5 (l236-c238), 6 (L276-i279), 7 (T319-A327), 8 (G354-l357) and 9 (P420-V421)], flanked by the remaining 11 helices [4 (Y145-S155), 5 (S167-A179), 6 (G193-l201), 7 (P207-q212), 8 (G219-S223), 9 (K228-G233), 10 (A250-K272), 11 (P301-K314), 12 (t337-l342), 13 (V363-W369) and 14 (D380-A402)].

Fig .4. Calculated root mean square deviation (RMSD) graph using GROMACS software. X-axis time (ps), y-axis RMSD (nm).

b Fig. 6. three dimensional structure of GPAt protein. Model (Ostreococcus lucimarinus) (a), template (Cucurbita moschata 1K30) (b). the modeled structure of GPAt protein in Ostreococcus lucimarinus shows similar topology and active site structure with the template. the monomer consists of 14 helices (1-14) and 9 (a-i) strands.

Fig. 5. calculated energy vs. time plot using GROMACS software. x-axis time (ps), y-axis total energy.

the structural analysis of 3-D structure of the built monomer of GPAt protein (Mol. wgt.: 472.733KDa; 429 Biotechnol. & Biotechnol. eq. 26/2012/1

A striking feature of GPAt proteins is the presence of a large cleft in domain ii. Multiple sequence alignment suggests that the invariant histidine and Aspartic residues, referenced as (h189) and (D194), respectively in the present model (Fig. 7) are separated by four less conserved residues in an h-X-X-X-X D configuration and located near the hollow tunnel shaped cleft region. Previous studies have established the crucial role 2797

of histidine residue in the h(X4) D consensus sequence in the binding of glycerol-3-phosphate moiety and catalysis of glycerolipid acyltransferase (14).

analogous to the templates e142 and D144 residues (Fig. 8). earlier report of site directed mutagenic study in Cucurbita moschata (squash) GPAT protein confirms K193, h194, R235 and R237 as the key catalytic residues involved in binding the negatively charged phosphate group of glycerol-3-phosphate (33), thus suggesting that the topological configuration of the present modeled structure resembles the crystal structure of its template.

Fig. 9. Ramachandran plot calculation for 3-D model of GPAT computed with PROCHECK.

Fig. 7. Schematic representation of the tunnel shaped cleft of domain ii of GPAt protein and the associated residues that play a vital role in the enzymes catalytic mechanism.

Fig. 10. ERRAT calculation of non-bonded interactions between different atom types of the modeled GPAt protein.

Fig. 8. Predicted structure of the glycerol-3-phosphate binding domain of Ostreococcus lucimarinus. the binding site lies in domain ii of the protein and the phosphate group of the G-3 P lies in a positively charged pocket (arrowed) formed by the side chains of K243, h244, R285 , and R287.

A cluster of positively charged conserved residues viz. h189, K243, h244, R285and R287 analogous to h139, K193, h194, R235, R237 residues of the template has been identified as the binding site for the phosphate group of glycerol-3-phosphate. these positively charged residues, flanking the H(X4) D motif of the cleft, are balanced by two acidic residues viz. e192 and D194 2798

Structural validation of the model Using various structure validation tools the quality of the monomeric structure of GPAt protein of O. lucimarinus was assessed. Analysis of the results of Ramachandran plot, suggest that the modeled structure has 92.5%, 6.5%, 0.8% and 0.3% of residues in core, additional allowed, generously allowed regions and disallowed regions, respectively (Fig. 9). the high scoring values obtained by various other protein validation tools viz., VERIfY 3D, ERRAT and PSQS (Fig. 10) indicate the overall accuracy of the developed model (Table 1). in addition the good structural and packing architecture as predicted through VADAR programme, conclusively confirms the reliability of the developed structure for further in silico studies (Table 2). Biotechnol. & Biotechnol. eq. 26/2012/1

Quality scores of modeled GPAT protein using different structure validation programmes Structure GPAT model
a b c d

TABLE 1

Core 92.5

PROCHECK a Additional Generously allowed allowed 6.5 0.8

Disallowed 0.3

VERIFY 3D b 63.49

ERRAT c 81.08

PSQS d -0.0908

percentage of residues with and conformational angles in the core, additional allowed, generously allowed and disallowed regions of Ramachandran Plot; percentage of residues with average 3D-1D score > 0.2; percentage of the protein for which the calculated error value falls below the 95% rejection limit; the average value for the PSQS scores of a representative set of PDB structures is -0.27 and most structures have a PSQS less than -0.1.

Structural characteristics and features of the model as predicted using VADAR 1.4 programme Structure Template(1K30_A) Model protein MHBD () 2.2 2.2 MHBE -1.8 -1.8 MHPh -64.4 --63.0 MHPs -38.6 -40.1 MCG+ -65.5 -65.3 MCG66.0 67.9 TASA () 16896.6 23881.5 MRV () 134.9 137.0

TABLE 2

TV () 48954.1 58791.5

MHBD- Mean hydrogen bond distance; MHBE-Mean hydrogen bond energy; MHPh- Mean Helix Phi; MHPs- Mean Helix Psi; MCG+ -Mean Chi Gauche+; MCG- -Mean Chi Gauche-; TASA- Total accessible surface area; MRV- Mean residue volume; TV- Total volume (packing)

Finally, the validated model of GPAT was submitted to PMDB (PMDB ID: PM0077512) and it has accepted the model with less than 3% stereochemical check failures.
a

active site region of the protein as predicted by CASTp server (Fig. 12). In our computational model the largest cavity with 69 amino acid residues, 2224.2 2 area and 8099.9 volume 3 was presumed to be the active site region of the protein.

Fig. 12. Solvent exposed active site residues representation of GPAT protein using CASTp server. The active site residues are shown in green colour.

Fig. 11. Secondary structure assignment: 1K30: A (template) (a), Predicted 3-D model of GPAT protein (target) (b); coil -helices (red), -sheets (green), turns or coils (yellow).

Secondary structural prediction and active site identification The overall secondary structural comparison between the modeled O. lucimarinus GPAT protein and X-ray crystallographic structure of GPAT from Cucurbita moschata reveals close homology among the secondary structure elements between the two (Fig. 11). The H(X4) D motif and the positively charged residues lining the cleft, responsible for the enzymes catalytic mechanism were observed to lie within the Biotechnol. & Biotechnol. Eq. 26/2012/1

This is the first known report on the structural and functional prediction of TAG biosynthetic enzyme, GPAT of Ostreococcus lucimarinus, a green microalga. The architectural features of the 3D model suggest the monomer consists of nine -strands and fourteen -helices organised in two domains. The predicted structure shows similar topology and active site structure with the crystal structure used as template in the present study. Multiple sequence alignment revealed the presence of the active site triad His-X-X-X-X-Asp strictly conserved in all proteins, which plays a critical role in the enzyme function. This is a characteristic signature among all glycerolipid acyltransferases. The positively charged residues viz. K243, H244, R285, and R287 flanking the cleft region of domain II represent the binding site for phosphate moiety of glycerol-3-phosphate. Further, the results from various structure validation tools, viz., PROCHECK, WHATCHECK, Ramachandran plot, Verify 3D, PSQS and ERRAT, conclusively confirmed the accuracy 2799

Conclusions

and reliability of the developed model. The availability of the developed GPAT model structure together with insights gained in the catalytic mechanism of the protein in the present study could serve as a valuable reference for biotechnological studies to optimize microalgal strains for enhanced biofuel production.

Acknowledgements

Financial support from Department of Biotechnology, Government of India to carry out the work is gratefully acknowledged. Thanks are due to Prof. B. K. Mishra, Director, IMMT for providing the necessary computational grid facilities and encouragement.

1. Altschul S.F., Gish W., Miller W., Myer E.W., Lipman D.J. (1990) J. Mol. Biol., 215, 403-410. 2. Berman H.M., Westbrook J., Feng Z., Gililand G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. (2000) Nucleic Acids Res., 28, 235-242. 3. Castrignano T., De Meo P.D., Cozzetto D., Talamo I.G., Tramontano A. (2006) Nucleic Acids Res., 34, 306-309. 4. Coleman R.A., Lee D.P. (2004) Prog. Lipid Res., 43, 134176. 5. Colovos C., Yeates T.O. (1993) Protein. Sci., 2, 15111519. 6. Courchesne N.M.D., Parisien A., Wang B., Lan C.Q. (2009) J. Biotechnol., 141, 31-41. 7. Darden T., York D., Pedersen L. (1993) J. Chem. Phys., 98, 10089-10092. 8. Dundas J., Ouyang Z., Tseng J., Binkowski A., Turpaz Y., Liang J. (2006) Nucleic Acids Res., 34, W116-W118. 9. Eisenberg D., Luthy R., Bowie J.U. (1997) Method. Enzymol., 277, 396-404. 10. Frishman D., Argos P. (1993) Protein, 23, 566-579. 11. Goldberg I.K., Cohen Z. (2011) Biochimie, 93, 91-100. 12. Guex N., Peitsch M.C. (1997) Electrophoresis, 18, 27142723. 13. Hall T.A. (1999) Nucleic Acid Symposium Series, 41, 9598. 14. Heath R.J., Rock C.O. (1998) J. Bacteriol., 180, 14251430. 15. Hess B., Bekker H., Berendsen H.J.C., Fraaije J.G.E.M. (1997) J .Comp. Chem., 18, 1463-1472. 16. Hess B., Kutzner C., vander Spoel D., Lindahl E. (2008) J .Chem. Theor. Comp., 4, 435-447. 17. Hooft R.W.W., Vriend G., Sander C., Abola E.E. (1996) Nature, 381, 272-292. 18. Hu Q., Sommerfeld M., Jarvis E., Ghirardi M., Posewitz M., Seibert M., Darzins A. (2008) Plant. J., 54, 621-639. 19. Jain R.K., Coffey M., Lai K., Kumar A., Mackenzie S.L. (2000) Biochem. Soc. Trans, 28, 959-960. 20. Jako C., Kumar A., Wei Y., Zou J., Barton D.L., Giblin E.M., Covello P.S., Taylor D.C. (2001) Plant. Physiol., 126, 861-874. 2800

REFERENCES

21. Jaroszewski L., Pawlowski K., Godzik A. (1998) J .Mol. Model., 4, 294-309. 22. Jorgensen W.L., Chandrasekhar J., Madura J.D., Impey R.W., Klein M.L. (1983) J. Chem. Phys., 79, 926935. 23. Lardizabal K., Ehffertz R., Levering C., Mai J., Pedroso M.C., Jury T., Aasen E., Gruys K., Bennett (2008) Plant. Physiol., 148, 89-96. 24. Laskowiski R.A., MacArthur M.V., Moss D.S., Thornton J.M. (1993) J. Appl. Cryst., 26, 283-291. 25. Lindahl E., Hess B., vander Spoel D. (2001) J. Mol. Model., 7, 306-317. 26. Mukhopadhyay A., Redding A.M., Rutherford B.J., Keasling J.D. (2008) Curr. Opin. Biotech., 19, 228-234. 27. Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin, T.E. (2004) J. Comput. Chem., 25, 1605-1612. 28. Pienkos P.T., Darzins A. (2009) Biofuels. Biprod. Bioref., 3, 431-440. 29. Radakovits R., Jinkerson R.E., Darzins A., Posewitz M.C. (2010) Eukaryote Cell, 9, 486-501. 30. Rodriguez-Moya M., Gonzalez R. (2010) Biofuels, 1, 291-310. 31. Sali A., Blundell T.L. (1993) J. Mol. Biol., 234, 779-815. 32. Scott S.A., Davey M.P., Dennis J.S., Horst I., Howe C.J., Lea-Smith D.J., Smith A.J. (2010) Curr. Opin. Biotech., 21, 277-286. 33. Slabas A.R., Kroon J.T.M., Scheirer T.P., Gilroy J.S., Hayman M., Rice D.W., Turnbull A.P., Rafferty J.B., Fawcett T. (2002) J. Biol .Chem., 277, 43918-43923. 34. Taylor D.C., Katavic V., Zou J., Mackenzie S.L., Keller W.A., Friesen W., Barton D.L., Pedersen K.K., Michael Giblin E. (2001) Mol. Breed., 8, 317-322. 35. Thompson J.D., Gibson T.J., Plewniak F., Higgins D.G. (1997) Nucleic Acids Res., 25, 4876-4882. 36. Turnbull A.P., Rafferty J.B., Sedelnikova S.E., Kroon J.T.M., Simon J.W., Fawcett T., Nishida I., Murata N., Rice D.W. (2001) Structure, 9, 347-353. 37. Wendel A.A., Lewin T.M., Coleman R.A. (2009) Biochim. Biophys. Acta., 1791, 501-506. 38. Wijffels R.H., Barbosa M.J. (2010) Science, 329, 796799. 39. Willard L., Ranjan A., Zhang H., Monzavi H., Boyko R.F., Sykes B.D., Wishart D.S. (2003) Nucleic Acids Res., 31, 3316-3319. 40. Zheng P., Allen W.B., Roesler K., Williams M.E., Zhang S., Li J., Glassman K., Ranch J., Nubel D., Solawetz W. (2008) Nat. Genet., 40, 367-372. 41. Zou J., Katavic V., Giblin E.M., Barton D.L., Mackenzie S.L., Keller W.A., Hu X., Taylor D.C. (1997) Plant Cell, 9, 909-923. Biotechnol. & Biotechnol. Eq. 26/2012/1