Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: Collectively, co- and post-translational processing of proteins translated in the chloroplast form the final, biologically-active mature products from gene expression. Frequently these processes are unpredictable from protein primary sequence derived from DNA sequence, are often essential, and represent a collection of enzymes with evolved polypeptide substrate specificity and associated structural features that make them unique hybrids of their prokaryotic and eukaryotic counterparts. Utilizing a collection of proven biochemical approaches and past accomplishments, this proposal provided structure/function relationships and in vivo functional significance for peptide deformylase (PDF), methionine aminopeptidase (MAP), and protein lysine methyltransferase (PKMT). Previous and existing biochemical and structural characterization of PDF and the PKMT, Rubisco LSMT, from this laboratory provided the rationale and justification for the approaches outlined in this proposal. Completion of the proposed studies provided unique structure/function relationships for co-and post-translational processing enzymes, and identification of polypeptide substrate specificity, and the mechanistic information that explains how they interact with all chloroplast-translated proteins. PARTICIPANTS: The PI supervised all aspects of the project which included one post-doctoral scholar and one full-time technician. TARGET AUDIENCES: The general scientific community involved in chloroplast-related protein processing was the target audience. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The findings from these studies resulted in the development of new approaches for the control of weeds through the use of peptide deformylase inhibitors and the construction of transgenic plants resistant to those inhibitors. Others interested in chloroplast protein processing benefited from identification of the mechanism that determines the N-termini of chloroplast-translated proteins.
Publications
- Whitney SM, Kane HJ, Houtz RL, & Sharwood RE (2009) Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2. Plant Physiology,149(4):1887-1895.
- Raunser, S., Magnani, R., Huang, Z., Houtz, R. L., Trievel, R. C., Penczek, P. A., and Walz, T. (2009). Rubisco in complex with Rubisco large subunit methyltransferase. PNAS , 106: 3160-3165.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Data were acquired which demonstrated the influence of protein domain regions in chloroplast-localized peptide deformylase on polypeptide substrate specificity. Experiments were completed that distinguished between processive and distributive methylation of histone polypeptides by the histone methyltransferase SET8. Complexes were generated between Rubisco and Rubisco methyltransferase that could be imaged using single particle electron microscopy. These results were communicated through annual meetings at the American Society for Plant Biology and the American Society for Biochemistry and Molecular Biology. PARTICIPANTS: Dr. Robert L. Houtz, PI/PD, Coordinator for all research projects and funding. Dr. Lynnette Dirk, Research Specialist, responsible for day-to-day laboratory operations, collaborative efforts, experimental design, execution, and interpretation. Dr. Roberta Magnani, Post-Doctoral Scholar, Specifically responsible for all of the research related to the dissection of alternative protein substrates for Rubisco LSMT. Ms. Amanda Hizer, Undergraduate Agricultural Biotechnology major, responsible for peptide deformylase enzyme assays and analyses as part of an undergraduate research project. Partner Organizations include the University of Michigan and the Harvard Medical School. Collaborators include, Dr. David Rodgers, Department of Biochemistry, University of Kentucky; Dr. Tom Waltz, Harvard Medical School; and Dr. Ray Trievel, the University of Michigan. TARGET AUDIENCES: General scientific community, particularly those involved in protein chemistry and structure/function relationships in enzymes. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Mechanisms which control peptide deformylase polypeptide substrate specificity were unknown until the completion and publication of our experiments on CD-loop swap regions between different forms of peptide deformylase. We provided the first structural analyses of a macromolecular complex between a SET-domain protein methyltransferase and its intact polypeptide substrate. The first kinetic manifestation for processive versus distributive methylation by a SET-domain protein methyltransferase was provided by this project. A detailed molecular mechanism was reported for the process used by the histone protein methyltransferase SET8 for site-specific methylation of a lysyl residue in histone H4.
Publications
- Couture, J. F., L. M. Dirk, J. S. Brunzelle, R. L. Houtz, and R. C. Trievel. 2008. Structural origins for the product specificity of SET domain protein methyltransferases. Proc.Natl.Acad.Sci.U.S.A. 105:20659-20664.
- Dirk, L. M., J. J. Schmidt, Y. Cai, J. C. Barnes, K. M. Hanger, N. R. Nayak, M. A. Williams, R. B. Grossman, R. L. Houtz, and D. W. Rodgers. 2008. Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture. Biochem.J 413:417-427.
- Houtz, R. L., R. Magnani, N. R. Nayak, and L. M. Dirk. 2008. Co- and post-translational modifications in Rubisco: unanswered questions. J Exp.Bot. 59:1635-1645.
- Dinkins, R. D., S. M. Majee, N. R. Nayak, D. Martin, Q. Xu, M. P. Belcastro, R. L. Houtz, C. M. Beach, and A. B. Downie. 2008. Changing transcriptional initiation sites and alternative 5'- and 3'-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments. Plant J 55:1-13.
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Progress 01/01/07 to 12/31/07
Outputs
Experiments were conducted to determine the kinetic reaction mechanism and polypeptide substrate specificity for Rubisco LSMT. The results were reported at seminars and national meetings.
Impacts
A model was developed for SET domain protein lysine methyltransferases in which initial binding of polypeptide substrate and S-adenosylmethionine is random, with polypeptide binding followed by deprotonation of the epsilon-amine of the target lysyl residue and subsequent methylation. Following methyl group transfer, S-adenosylhomocysteine and monomethylated polypeptide dissociate from monomethyltransferases, but di- and trimethyltransferases begin a successive and catalytically obligatory deprotonation of enzyme-bound methylated lysyl intermediates, which along with binding and release of S-adenosylmethionine and S-adenosylhomocysteine is manifested as a hybrid ping-pong-like reaction mechanism. Additionally, The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were determined. The polypeptide consensus recognition sequence was represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-Z, where X is any residue, Lys is
the methylation site, and Z is any aromatic or hydrophobic residue.
Publications
- Magnani,R., N.R.Nayak, M.Mazarei, L.M.Dirk, and R.L.Houtz. 2007. Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase. J. Biol. Chem. 282:27857-27864.
- Dirk,L.M., E.M.Flynn, K.Dietzel, J.F.Couture, R.C.Trievel, and R.L.Houtz. 2007. Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases. Biochemistry 46:3905-3915.
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Progress 01/01/06 to 12/31/06
Outputs
The kinetic reaction mechanism and processive versus distributive methyl group transfer was determined for pea Rubisco large subunit methyltransferase (PsLSMT), a SET domain protein lysine methyltransferase (PKMT) catalyzing the formation of trimethyllysine-14 (Me3Lys-14) in the LS of Rubisco. Catalytically competent complexes between a PVDF-immobilized form of des(methyl) Rubisco and PsLSMT were used to demonstrate an absence of disassociation of PsLSMT from Rubisco after formation of Me- or Me2Lys, but release coincident with formation of Me3Lys. Catalytic rate constants determined for formation of Me3Lys were considerably less (10-fold) than rate constants determined for total radiolabel incorporation from [3H-methyl] S-adenosylmethionine (AdoMet). Kinetic reaction mechanism analyses under catalytic conditions favoring MeLys indicated a random or ordered reaction mechanism, while conditions favoring Me3Lys indicated a ping-pong mechanism. These results were
compared with kinetic reaction mechanism data and product analyses obtained for HsSET7/9 and SpCLR4, a mono- and dimethyltransferase respectively, and suggest a predictive ability of kinetic reaction mechanism analyses for single versus multiple methyl group transfers by SET domain PKMTs. A model is proposed for SET domain PKMTs where initial binding of polypeptide substrate and AdoMet is random, with polypeptide binding followed by deprotonation of the epsilon-amine of the target lysyl residue and subsequent methylation. Following methyl group transfer, S-adenosylhomocysteine (AdoHcy) and monomethylated polypeptide disassociate from monomethyltransferases, but di- and trimethyltransferases begin a successive and catalytically obligatory deprotonation of enzyme bound methylated lysyl intermediates, which along with binding and release of AdoMet and AdoHcy, is manifested as a ping-pong reaction mechanism. The polypeptide substrate specificity determinants for pea Rubisco large subunit
methyltransferase were investigated using a fusion protein construct between the N-termini of the large subunit of Rubisco and human carbonic anhydrase II. A total of 38 conservative and non-conservative amino acid substitutions were engineered in the Rubisco LSMT: HCA II fusion protein spanning the three positions (P-3 to P+3) flanking the target Lys-14 methylation site. The catalytic efficiency (kcat/Km) of Rubisco LSMT was determined for each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X - G/S - F/Y - K - A/K/R - G/S - U where X is any residue, K is the methylation site, and U is any aromatic or hydrophobic residue was used to predict potential alternative substrates for Rubisco LSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase. In vitro methylation assays using a bacterially expressed form of gamma-tocopherol methyltransferase from
Arabidopsis and Rubisco LSMT confirmed recognition and methylation of gamma-tocopherol methyltransferase by Rubisco LSMT.
Impacts
Research on enzymes specific to and essential in plants has resulted in identification of novel mechanisms regulating gene expression, viable alternatives to antibiotic resistance based selectable markers in plant transformation vectors, and new compounds with broad-spectrum herbicidal activity.
Publications
- Dirk, L. M. A., Trievel, R. C., and Houtz, R. L. (2006) Non-Histone Protein Methyltransferases: Structure and Catalytic Roles, in The Enzymes (Tamanoi, F. and Clarke, S. eds) Vol. 24 pp. 179-229, Elsevier Academic Press, Burlington, MA
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Progress 01/01/05 to 12/31/05
Outputs
Research has resulted in the characterization of chloroplast-localized post- and co-translational protein processing enzymes. Current efforts are directed towards the identification of alternative protein substrates and elucidation of protein specificity determinants for Rubisco LSMT. An artificial protein substrate for Rubisco LSMT, consisting of a fusion protein between the first 25 amino acid residues from the N-terminus of the LS and the N-terminus of human carbonic anhydrase, is being used to map protein specificity determinants through site-directed mutagenesis of residues known to occupy the polypeptide binding cleft in Rubisco LSMT from Val11 to Val17 in the LS. Preliminary results indicate considerable flexibility in several positions but strict requirements for aromatic side-chain residues at position 13 and Gly at position 12. Collectively the results suggest a number of other proteins as potential substrates for Rubisco LSMT. As an additional effort to
identify alternative polypeptide substrates for Rubisco LSMT, as well as potential functional significance of lysine methylation, a mouse orthologue of Rubisco LSMT was used to probe a human protein macroarray. The results identified at least three potential polypeptide substrates. Chloroplast-localized peptide deformylase (DEF) has been shown to be an essential co-translational processing enzyme in plants. Current evidence suggests that the lethality of DEF inhibition is a consequence of disruption of PSII complex assembly and inhibition of D1 polypeptide turnover or translation. The recent acquisition of an X-ray crystal structure clearly identifies a substrate binding cleft unique to plant DEF that may contain the specificity determinants for the newly translated D1 polypeptide N-terminus. The exact nature of the residues in this cleft are being identified through molecular modeling refinement.
Impacts
Research on enzymes specific to and essential in plants has resulted in identification of novel mechanisms regulating gene expression, viable alternatives to antibiotic resistance based selectable markers in plant transformation vectors, and new compounds with broad-spectrum herbicidal activity.
Publications
- Dirk, L.M.A., Trievel, R.C., Houtz, R.L. 2005. Non-Histone Protein Lysine Methyltransferases - structure and catalytic roles. In Fuyu Tamanoi and Steven Clarke (eds.), Protein Methyltransferases. The Enzymes, Academic Press (in press).
- Shepherd, R. W., Bass, W.T., Houtz, R.L. and Wagner, G.J. 2005. Phylloplanins of tobacco are defensive proteins deployed on aerial surfaces by short glandular trichomes. Plant Cell 17:1851-1861.
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Progress 01/01/04 to 12/31/04
Outputs
Research in my laboratory is focused on characterization of chloroplast-localized post- and co-translational protein processing enzymes with emphasis on the large (LS) and small (SS) subunits of Rubisco. A recent examination of structure/function relationships in pea Rubisco LS N-methyltransferase (Rubisco LSMT) with emphasis on the highly conserved protein motif known as the SET domain, provided novel information identifying the mechanism for recognition and subsequent methylation of target protein lysyl residues by other protein N-methyltransferases. In Rubisco a conserved amino acid sequence in the N-terminal region of the LS from Ala-9 to Lys-14 establishes the specificity for methylation at Lys-14. This region fits into a narrow cleft in Rubisco LSMT, with specificity primarily for small side chain amino acids with the exception of Phe-13, whose side chain resides in a deep hydrophobic cleft. The structure suggests that there could be other protein substrates for
Rubisco LSMT with sequence motifs homologous to the region in the LS of Rubisco. We have recently engineered an alternate substrate for Rubisco LSMT by fusing the N-terminal sequence from the LS to the N-termini of human carbonic anhydrase (CA). The LS:CA fusion protein gives us the opportunity to explore the specificity, and moreover any potential flexibility in the specificity, between Rubisco LSMT and the N-terminal region of the LS. Identification of alternate sequence motifs which are capable of methylation may also result in the identification of alternative substrates. Studies have been completed which have unambiguously identified plant peptide deformylases DEF1 and DEF2 as the targets of the inhibitor actinonin, and that complete functional redundancy exist between DEF1 and DEF2. Over-expression of either DEF1 or DEF2 in transgenic tobacco plants resulted in complete immunity from the toxicity of actinonin. Other studies confirmed that the effects of actinonin are from an
inhibition of D1 polypeptide translation or turnover, and that one of the earliest effects is a disassembly of the photosystem II complex.
Impacts
Information about the specificity of Rubisco LSMT will identify other protein substrates. Identification of peptide deformylase as the target of peptide deformylase inhibitors opens possibilities for the development of a new class of broad-spectrum herbicides and the ability to engineer crop resistance.
Publications
- No publications reported this period
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