Publications by authors named "Lynnette M A Dirk"

28 Publications

  • Page 1 of 1

ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth.

Plant J 2020 09 29;104(1):268-282. Epub 2020 Jul 29.

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China.

Seed aging tolerance and rapid seedling growth are important agronomic traits for crop production; however, how these traits are controlled at the molecular level remains largely unknown. The unaged seeds of two independent maize DEHYDRATION-RESPONSIVE ELEMENT-BINDING2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2 (ZmGH3.2, encoding indole-3-acetic acid [IAA] deactivating enzyme), and increased IAA in their embryo, produced longer seedling shoots and roots, than the null segregant (NS) controls. However, the zmdreb2a seeds, with decreased expression of RAFFINOSE SYNTHASE (ZmRAFS) and less raffinose in their embryo, exhibit decreased seed aging tolerance, than the NS controls. Overexpression of ZmDREB2A in maize protoplasts increased the expression of ZmGH3.2, ZmRAFS genes and that of a Rennila LUCIFERASE reporter (Rluc) gene, which was controlled by either the ZmGH3.2- or ZmRAFS-promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assay quantitative polymerase chain reaction showed that ZmDREB2A directly binds to the DRE motif of the promoters of both ZmGH3.2 and ZmRAFS. Exogenous supplementation of IAA to the unaged, germinating NS seeds increased subsequent seedling growth making them similar to the zmdreb2a seedlings from unaged seeds. These findings provide evidence that ZmDREB2A regulates the longevity of maize seed by stimulating the production of raffinose while simultaneously acting to limit auxin-mediated cell expansion.
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http://dx.doi.org/10.1111/tpj.14922DOI Listing
September 2020

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Plant Cell 2020 09 9;32(9):2898-2916. Epub 2020 Jul 9.

Research School of Biology, The Australian National University, Acton, Australian Capital Territory 2601, Australia

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast gene and nuclear genes. Here we develop an RNAi- tobacco () master-line, tobRrΔS, for producing homogenous plant Rubisco by L-S operon chloroplast transformation. Four genotypes encoding alternative genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a gene whose codon use and 5' untranslated-region matched Additional tobacco genotypes produced here incorporated differing potato () - operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pS-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pS However, the βA-βB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pS-subunit, which reduced CE and CO/O specificity 40% and 15%, respectively. Returning the gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.
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http://dx.doi.org/10.1105/tpc.20.00288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474299PMC
September 2020

Late Embryogenesis Abundant Protein-Client Protein Interactions.

Plants (Basel) 2020 Jun 29;9(7). Epub 2020 Jun 29.

Department of Horticulture, University of Kentucky Seed Biology Program, Plant Science Building, 1405 Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA.

The intrinsically disordered proteins belonging to the LATE EMBRYOGENESIS ABUNDANT protein (LEAP) family have been ascribed a protective function over an array of intracellular components. We focus on how LEAPs may protect a stress-susceptible proteome. These examples include instances of LEAPs providing a shield molecule function, possibly by instigating liquid-liquid phase separations. Some LEAPs bind directly to their client proteins, exerting a holdase-type chaperonin function. Finally, instances of LEAP-client protein interactions have been documented, where the LEAP modulates (interferes with) the function of the client protein, acting as a surreptitious rheostat of cellular homeostasis. From the examples identified to date, it is apparent that client protein modulation also serves to mitigate stress. While some LEAPs can physically bind and protect client proteins, some apparently bind to assist the degradation of the client proteins with which they associate. Documented instances of LEAP-client protein binding, even in the absence of stress, brings to the fore the necessity of identifying how the LEAPs are degraded post-stress to render them innocuous, a first step in understanding how the cell regulates their abundance.
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http://dx.doi.org/10.3390/plants9070814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412488PMC
June 2020

Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and plants.

J Biol Chem 2020 06 4;295(23):8064-8077. Epub 2020 May 4.

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear. In this study, we report that expression of from maize (or corn, ) () is induced by drought, heat, cold, and salinity stresses. We found that mutant maize plants completely lack raffinose and hyper-accumulate galactinol and are more sensitive to drought stress than the corresponding null-segregant (NS) plants. This indicated that ZmRAFS and its product raffinose contribute to plant drought tolerance. overexpression in enhanced drought stress tolerance by increasing -inositol levels via ZmRAFS-mediated galactinol hydrolysis in the leaves due to sucrose insufficiency in leaf cells and also enhanced raffinose synthesis in the seeds. Supplementation of sucrose to detached leaves converted ZmRAFS from hydrolyzing galactinol to synthesizing raffinose. Taken together, we demonstrate that ZmRAFS enhances plant drought tolerance through either raffinose synthesis or galactinol hydrolysis, depending on sucrose availability in plant cells. These results provide new avenues to improve plant drought stress tolerance through manipulation of the raffinose anabolic pathway.
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http://dx.doi.org/10.1074/jbc.RA120.013948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278351PMC
June 2020

ZmDREB1A Regulates RAFFINOSE SYNTHASE Controlling Raffinose Accumulation and Plant Chilling Stress Tolerance in Maize.

Plant Cell Physiol 2020 Feb;61(2):331-341

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.

Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. In this study, we report that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation was significantly decreased compared with controls after chilling stress. A similar characterization of the maize dehydration responsive element (DRE)-binding protein 1A mutant (zmdreb1a) showed that ZmRAFS expression and raffinose content were significantly decreased compared with its control under chilling stress. Overexpression of maize ZmDREB1A in maize leaf protoplasts increased ZmDREB1A amounts, which consequently upregulated the expression of maize ZmRAFS and the Renilla LUCIFERASE (Rluc), which was controlled by the ZmRAFS promoter. Deletion of the single dehydration-responsive element (DRE) in the ZmRAFS promoter abolished ZmDREB1A's influence on Rluc expression, while addition of three copies of the DRE in the ZmRAFS promoter dramatically increased Rluc expression when ZmDREB1A was simultaneously overexpressed. Electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative PCR demonstrated that ZmDREB1A directly binds to the DRE motif in the promoter of ZmRAFS both in vitro and in vivo. These data demonstrate that ZmRAFS, which was directly regulated by ZmDREB1A, enhances both raffinose biosynthesis and plant chilling stress tolerance.
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http://dx.doi.org/10.1093/pcp/pcz200DOI Listing
February 2020

Maize HSFA2 and HSBP2 antagonistically modulate raffinose biosynthesis and heat tolerance in Arabidopsis.

Plant J 2019 10 12;100(1):128-142. Epub 2019 Jul 12.

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China.

Raffinose is thought to play an important role in plant tolerance of abiotic stress. We report here that maize HEAT SHOCK FACTOR A2 (ZmHSFA2) and HEAT SHOCK BINDING PROTEIN 2 (ZmHSBP2) physically interact with each other and antagonistically modulate expression of GALACTINOL SYNTHASE2 (ZmGOLS2) and raffinose biosynthesis in transformed maize protoplasts and Arabidopsis plants. Overexpression of ZmHSFA2 in Arabidopsis increased the expression of Arabidopsis AtGOLS1, AtGOLS2 and AtRS5 (RAFFINOSE SYNTHASE), increased the raffinose content in leaves and enhanced plant heat stress tolerance. Contrary to ZmHSFA2, overexpression of ZmHSBP2 in Arabidopsis decreased expression of AtGOLS1, AtGOLS2 and AtRS5, decreased the raffinose content in leaves and reduced plant heat stress tolerance. ZmHSFA2 and ZmHSBP2 also interact with their Arabidopsis counterparts AtHSBP and AtHSFA2 as determined using bimolecular fluorescence complementation assays. Furthermore, endogenous ZmHSBP2 and Rluc, controlled by the ZmHSBP2 promoter, are transcriptionally activated by ZmHSFA2 and inhibited by ZmHSBP2 in maize protoplasts. These findings provide insights into the transcriptional regulation of raffinose biosynthetic genes, and the tolerance their product confers to plant heat stress.
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http://dx.doi.org/10.1111/tpj.14434DOI Listing
October 2019

Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate GALACTINOL SYNTHASE2 Expression Controlling Seed Raffinose Accumulation.

J Agric Food Chem 2019 Apr 5;67(15):4214-4223. Epub 2019 Apr 5.

Department of Horticulture, Seed Biology, College of Agriculture, Food, and Environment , University of Kentucky , Lexington , Kentucky 40546 , United States.

Raffinose, an oligosaccharide found in many seeds, plays an important role in seed vigor; however, the regulatory mechanism governing raffinose biosynthesis remains unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1 ( zmvp1) mutant seeds, start accumulating galactinol and raffinose 28 days after pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the expression of GALACTINOL SYNTHASE2 ( GOLS2) was down-regulated relative to WT. Further experiments showed that the expression of ZmGOLS2 was up-regulated by ZmABI5 but not by ZmVP1, and it was further increased by the coexpression of ZmABI5 and ZmVP1 in maize protoplasts. ZmABI5 interacted with ZmVP1, while ZmABI5, but not ZmVP1, directly binds to the ZmGOLS2 promoter. Together, all of the findings suggest that ZmVP1 interacts with ZmABI5 and regulates ZmGOLS2 expression and raffinose accumulation in maize seeds.
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http://dx.doi.org/10.1021/acs.jafc.9b00322DOI Listing
April 2019

PHYTOCHROME INTERACTING FACTOR1 interactions leading to the completion or prolongation of seed germination.

Plant Signal Behav 2018 8;13(10):e1525999. Epub 2018 Oct 8.

a Department of Horticulture, Seed Biology Group , University of Kentucky , Lexington , KY , USA.

In Arabidopsis thaliana, the basic Helix Loop Helix transcription factor, PHYTOCHROME INTERACTING FACTOR1 (PIF1) is known to orchestrate the seed transcriptome such that, ultimately, proteins repressing the completion of germination are produced in darkness. While PIF1-mediated control of abscisic acid (ABA) and gibberellic acid (GA) anabolism/catabolism is indirect, PIF1 action favors ABA while discriminating against GA, firmly establishing ABA's repressive influence on the completion of germination. The result is tissue that is more sensitive to and producing more ABA; and is less responsive to and deficient in GA. Illumination of the appropriate wavelength activates phytochrome which enters the nucleus, and binds to PIF1, initiating PIF1's phosphorylation by diverse kinases, subsequent polyubiquitination, and hydrolysis. One mechanism by which phosphorylated PIF1 is eliminated from the cells of the seed upon illumination involves an F-BOX protein, COLD TEMPERATURE GERMINATING10 (CTG10). Discovered in an unbiased screen of activation tagged lines hastening the completion of seed germination at 10°C, one indirect consequence of CTG10 action in reducing PIF1 titer, should be to enhance the transcription of genes whose products work to increase bioactive GA titer, shifting the intracellular milieu from one that is repressive to, toward one conducive to, the completion of seed germination. We have tested this hypothesis using a variety of Arabidopsis lines altered in CTG10 amounts. Here we demonstrate using bimolecular fluorescence complementation that PIF1 interacts with CTG10 and show that, in light exposed seeds, PIF1 is more persistent in ctg10 relative to WT seeds while it is less stable in seeds over-expressing CTG10. These results are congruent with the relative transcript abundance from three genes whose products are involved in bioactive GA accumulation. We put forth a model of how PIF1 interactions in imbibed seeds change during germination and how a permissive light signal influences these changes, leading to the completion of germination of these positively photoblastic propagules.
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http://dx.doi.org/10.1080/15592324.2018.1525999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204810PMC
June 2019

KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1.

Proc Natl Acad Sci U S A 2018 04 9;115(17):E4120-E4129. Epub 2018 Apr 9.

Department of Horticulture, Seed Biology, University of Kentucky, Lexington, KY 40546;

Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination. is expressed predominantly in the hypocotyl, and the protein is localized to the nucleus. CTG10 interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) and helps regulate its abundance accelerates the loss of PIF1 in light, increasing germination efficiency, while lines fail to complete germination in darkness, which is reversed by concurrent - Double-mutant () lines demonstrated that PIF1 is epistatic to CTG10. Both CTG10 and PIF1 amounts decline during seed germination in the light but reaccumulate in the dark. PIF1 in turn down-regulates transcription, suggesting a feedback loop of CTG10/PIF1 control. The genetic, physiological, and biochemical evidence, when taken together, leads us to propose that PIF1 and CTG10 coexist, and even accumulate, in the nucleus in darkness, but that, following illumination, CTG10 assists in reducing PIF1 amounts, thus promoting the completion of seed germination and subsequent seedling development.
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http://dx.doi.org/10.1073/pnas.1711919115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924874PMC
April 2018

Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana.

Mol Plant 2017 12 7;10(12):1540-1555. Epub 2017 Nov 7.

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China; The Key Laboratory of Biology and Genetics Improvement of Maize in Arid Area of Northwest Region, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address:

Raffinose family oligosaccharides (RFOs) accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE (GOLS), RAFFINOSE SYNTHASE (RS), and STACHYOSE SYNTHASE (STS) are the enzymes responsible for RFO biosynthesis in plants. Interestingly, only raffinose is detected in maize seeds, and a unique maize RS gene (ZmRS) was identified. In this study, we found that two independent mutator (Mu)-interrupted zmrs lines, containing no raffinose but hyperaccumulating galactinol, have significantly reduced seed vigor, compared with null segregant controls. Unlike maize, Arabidopsis thaliana seeds contain several RFOs (raffinose, stachyose, and verbascose). Manipulation of A. thaliana RFO content by overexpressing ZmGOLS2, ZmRS, or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS, or overexpression of ZmGOLS2 alone, significantly increased the total content of RFOs and enhanced Arabidopsis seed vigor. Surprisingly, while overexpression of ZmRS increased seed raffinose content, its overexpression dramatically decreased seed vigor and reduced the seed amounts of galactinol, stachyose, and verbascose. In contrast, the atrs5 mutant seeds are similar to those of the wild type with regard to seed vigor and RFO content, except for stachyose, which accumulated in atrs5 seeds. Total RFOs, RFO/sucrose ratio, but not absolute individual RFO amounts, positively correlated with A. thaliana seed vigor, to which stachyose and verbascose contribute more than raffinose. Taken together, these results provide new insights into regulatory mechanisms of seed vigor and reveal distinct requirement for RFOs in modulating seed vigor in a monocot and a dicot.
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http://dx.doi.org/10.1016/j.molp.2017.10.014DOI Listing
December 2017

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7.

J Biol Chem 2016 07 27;291(31):16240-8. Epub 2016 May 27.

From the Gladstone Institute of Virology and Immunology, San Francisco, California 94158, Departments of Medicine and

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat.
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http://dx.doi.org/10.1074/jbc.M116.735415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965572PMC
July 2016

ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A.

Plant Mol Biol 2016 Jan 19;90(1-2):157-70. Epub 2015 Nov 19.

State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling, 712100, Shaanxi, China.

GALACTINOL SYNTHASE is the first committed enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize (Zea mays) GALACTINOL SYNTHASE2 gene (ZmGOLS2) as abiotic stress induced. To further investigate the regulation of ZmGOLS2 gene expression, individual luciferase expression vectors,in which the luciferase gene was controlled by different lengths of the ZmGOLS2 promoter, were co-transfected into maize protoplasts with either a ZmDREB2A- or a GFP-expression vector. Over-expression of ZmDREB2A up-regulated both the expression of the luciferase gene controlled by the ZmGOLS2 promoter and the endogenous ZmGOLS2 gene in protoplasts. Only one of the two DRE elements in the ZmGOLS2 promoter was identified as necessary for this up-regulation. Expression vectors of GFP, ZmGOLS2 or ZmDREB2A were stably transformed into Arabidopsis. Expression of ZmDREB2A up-regulated the AtGOLS3 gene but only over-expression of ZmGOLS2 resulted in hyper-accumulation of galactinol and raffinose. Regardless, under drought-, heat shock-, high osmotic- or salinity-stress conditions, both the ZmGOLS2- and the ZmDREB2A- expressing plants had greater germination percentages, greater percentages of seedlings becoming autotropic, and/or greater survival percentages during/after stress than the control plants. Under normal growing conditions, transgenic Arabidopsis plants expressing the ZmGOLS2 gene had similar growth to that of untransformed wild type or GFP-expressing control plants, whereas ZmDREB2A over-expressing plants exhibited retarded growth relative to either of the controls. These data suggest that over-expression of ZmGOLS2, rather than the transcription factor ZmDREB2A, is a more practical target for generation of abiotic-stress tolerant crops.
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http://dx.doi.org/10.1007/s11103-015-0403-1DOI Listing
January 2016

Manipulating unconventional CH-based hydrogen bonding in a methyltransferase via noncanonical amino acid mutagenesis.

ACS Chem Biol 2014 Aug 3;9(8):1692-7. Epub 2014 Jul 3.

Howard Hughes Medical Institute , Ann Arbor, Michigan 48109, United States.

Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chemistry calculations to predict the mutation's effects, coupled with biochemical and structural studies, we observed that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.
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http://dx.doi.org/10.1021/cb5001185DOI Listing
August 2014

Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

J Am Chem Soc 2013 Oct 7;135(41):15536-48. Epub 2013 Oct 7.

Howard Hughes Medical Institute , Ann Arbor, Michigan 48109, United States.

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.
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http://dx.doi.org/10.1021/ja407140kDOI Listing
October 2013

Calmodulin methyltransferase is an evolutionarily conserved enzyme that trimethylates Lys-115 in calmodulin.

Nat Commun 2010 Jul 27;1:43. Epub 2010 Jul 27.

Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546, USA.

Calmodulin (CaM) is a key mediator of calcium-dependent signalling and is subject to regulatory post-translational modifications, including trimethylation of Lys-115. In this paper, we identify a class I, non-SET domain protein methyltransferase, calmodulin-lysine N-methyltransferase (EC 2.1.1.60). A polypeptide chosen from a fraction enriched in calmodulin methyltransferase activity was trypsinized and analysed by tandem mass spectrometry. The amino-acid sequence obtained identified conserved, homologous proteins of unknown function across a wide range of species, thus implicating a broad role for lysine methylation in calcium-dependent signalling. Encoded by c2orf34, the human homologue is a component of two related multigene deletion syndromes in humans. Human, rat, frog, insect and plant homologues were cloned and Escherichia coli-recombinant proteins catalysed the formation of a trimethyllysyl residue at position 115 in CaM, as verified by product analyses and mass spectrometry.
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http://dx.doi.org/10.1038/ncomms1044DOI Listing
July 2010

SET7/9 catalytic mutants reveal the role of active site water molecules in lysine multiple methylation.

J Biol Chem 2010 Oct 1;285(41):31849-58. Epub 2010 Aug 1.

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.
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http://dx.doi.org/10.1074/jbc.M110.114587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951256PMC
October 2010

Structural origins for the product specificity of SET domain protein methyltransferases.

Proc Natl Acad Sci U S A 2008 Dec 16;105(52):20659-64. Epub 2008 Dec 16.

Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

SET domain protein lysine methyltransferases (PKMTs) regulate transcription and other cellular functions through site-specific methylation of histones and other substrates. PKMTs catalyze the formation of monomethylated, dimethylated, or trimethylated products, establishing an additional hierarchy with respect to methyllysine recognition in signaling. Biochemical studies of PKMTs have identified a conserved position within their active sites, the Phe/Tyr switch, that governs their respective product specificities. To elucidate the mechanism underlying this switch, we have characterized a Phe/Tyr switch mutant of the histone H4 Lys-20 (H4K20) methyltransferase SET8, which alters its specificity from a monomethyltransferase to a dimethyltransferase. The crystal structures of the SET8 Y334F mutant bound to histone H4 peptides bearing unmodified, monomethyl, and dimethyl Lys-20 reveal that the phenylalanine substitution attenuates hydrogen bonding to a structurally conserved water molecule adjacent to the Phe/Tyr switch, facilitating its dissociation. The additional space generated by the solvent's dissociation enables the monomethyllysyl side chain to adopt a conformation that is catalytically competent for dimethylation and furnishes sufficient volume to accommodate the dimethyl epsilon-ammonium product. Collectively, these results indicate that the Phe/Tyr switch regulates product specificity through altering the affinity of an active-site water molecule whose dissociation is required for lysine multiple methylation.
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http://dx.doi.org/10.1073/pnas.0806712105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634886PMC
December 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 2008 Aug;413(3):417-27

Plant Physiology/Biochemistry/Molecular Biology Program, Department of Horticulture, University of Kentucky, 441 Plant Science Building, Lexington, KY 40546-0312, USA.

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.
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http://dx.doi.org/10.1042/BJ20071641DOI Listing
August 2008

Co- and post-translational modifications in Rubisco: unanswered questions.

J Exp Bot 2008 18;59(7):1635-45. Epub 2008 Mar 18.

Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, 1405 Veterans Drive, 441 Plant Science Building, Lexington, KY 40546-0312, USA.

Both the large (LS) and small (SS) subunits of Rubisco are subject to a plethora of co- and post-translational modifications. With the exceptions of LS carbamylation and SS transit sequence processing, the remaining modifications, including deformylation, acetylation, methylation, and N-terminal proteolytic processing of the LS, are still biochemically and/or functionally undefined although they are found in nearly all forms of Rubisco from vascular plants. A collection of relatively unique enzymes catalyse these modifications, and several have been characterized in other organisms. Some of the observed modifications in the LS and SS clearly suggest novel changes in enzyme specificity and/or activity, and others have common features with other co- and post-translationally modifying enzymes. With the possible exception of Lys14 methylation in the LS, processing of both the LS and SS of Rubisco is by default an ordered process sequentially leading up to the final forms observed in the holoenzyme. An overview of the nature of structural modifications in the LS and SS of Rubisco is presented, and, where possible, the nature of the enzymes catalysing these modifications (either through similarity with other known enzymes or through direct enzymological characterization) is described. Overall, there are a distinct lack of functional and mechanistic observations for modifications in Rubisco and thus represent many potentially productive avenues for research.
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http://dx.doi.org/10.1093/jxb/erm360DOI Listing
October 2008

Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase.

J Biol Chem 2007 Sep 17;282(38):27857-64. Epub 2007 Jul 17.

Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546-0312, USA.

Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.
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http://dx.doi.org/10.1074/jbc.M702069200DOI Listing
September 2007

Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases.

Biochemistry 2007 Mar 6;46(12):3905-15. Epub 2007 Mar 6.

Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, 401D Plant Science Building, University of Kentucky, Lexington, Kentucky 40546-0312, USA.

Processive versus distributive methyl group transfer was assessed for pea Rubisco large subunit methyltransferase, a SET domain protein lysine methyltransferase catalyzing the formation of trimethyllysine-14 in the large subunit of Rubisco. Catalytically competent complexes between an immobilized form of des(methyl) Rubisco and Rubisco large subunit methyltransferase were used to demonstrate enzyme release that was co-incident with and dependent on formation of trimethyllysine. Catalytic rate constants determined for formation of trimethyllysine were considerably lower ( approximately 10-fold) than rate constants determined for total radiolabel incorporation from [3H-methyl]-S-adenosylmethionine. Double-reciprocal velocity plots under catalytic conditions favoring monomethyllysine indicated a random or ordered reaction mechanism, while conditions favoring trimethyllysine suggested a hybrid ping-pong mechanism. These results were compared with double-reciprocal velocity plots and product analyses obtained for HsSET7/9 (a monomethyltransferase) and SpCLR4 (a dimethyltransferase) and suggest a predictive ability of double-reciprocal velocity plots for single versus multiple methyl group transfers by SET domain protein lysine methyltransferases. A model is proposed 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.
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http://dx.doi.org/10.1021/bi6023644DOI Listing
March 2007

Plant peptide deformylase: a novel selectable marker and herbicide target based on essential cotranslational chloroplast protein processing.

Plant Biotechnol J 2007 Mar;5(2):275-81

Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, KY 40546-0091, USA.

Transgenic tobacco plants expressing three different forms of Arabidopsis plant peptide deformylase (AtDEF1.1, AtDEF1.2 and AtDEF2; EC 3.5.1.88) were evaluated for resistance to actinonin, a naturally occurring peptide deformylase inhibitor. Over-expression of either AtDEF1.2 or AtDEF2 resulted in resistance to actinonin, but over-expression of AtDEF1.1 did not. Immunological analyses demonstrated that AtDEF1.2 and AtDEF2 enzymes were present in both stromal and thylakoid fractions in chloroplasts, but AtDEF1.1 was localized to mitochondria. The highest enzyme activity was associated with stromal AtDEF2, which was approximately 180-fold greater than the level of endogenous activity in the host plant. Resistance to actinonin cosegregated with kanamycin resistance in Atdef1.2-D and Atdef2-D transgenic plants. Here, we demonstrate that the combination of plant peptide deformylase and peptide deformylase inhibitors may represent a native gene selectable marker system for chloroplast and nuclear transformation vectors, and also suggest plant peptide deformylase as a potential broad-spectrum herbicide target.
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http://dx.doi.org/10.1111/j.1467-7652.2007.00238.xDOI Listing
March 2007

7 Non-histone protein lysine methyltransferases: Structure and catalytic roles.

Enzymes 2006 4;24:179-228. Epub 2007 Jun 4.

Department of Horticulture University of Kentucky 407 Plant Science Building Lexington, KY 40546, USA.

Non-histone protein lysine methyltransferases (PKMTs) represent an exceptionally diverse and large group of PKMTs. Even accepting the possibility of multiple protein substrates, if the number of different proteins with methylated lysyl residues and the number of residues modified is indicative of individual PKMTs there are well over a hundred uncharacterized PKMTs. Astoundingly, only a handful of PKMTs have been studied, and of these only a few with identifiable and well-characterized structure and biochemical properties. Four representative PKMTs responsible for trimethyllysyl residues in ribosomal protein LI 1, calmodulin, cytochrome c, and Rubisco are herein examined for enzymological properties, polypeptide substrate specificity, functional significance, and structural characteristics. Although representative of non-histone PKMTs, and enzymes for whichcollectively there is a large amount of information, individually each of the PKMTs discussed in this chapter suffers from a lack of at least some critical information. Other than the obvious commonality in the AdoMet substrate cofactor and methyl group transfer, these enzymes do not have common structural features, polypeptide substrate specificity, or protein sequence. However, there may be a commonality that supports the hypothesis that methylated lysyl residues act as global determinants regulating specific protein-protein interactions.
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http://dx.doi.org/10.1016/S1874-6047(06)80009-0DOI Listing
January 2016

Inhibition of peptide deformylase in Nicotiana tabacum leads to decreased D1 protein accumulation, ultimately resulting in a reduction of photosystem II complexes.

Am J Bot 2004 Sep;91(9):1304-11

Department of Horticulture, Agricultural Science Center North, University of Kentucky, Lexington, Kentucky 40546 USA;

Eukaryotic homologs of bacterial peptide deformylases were recently found in several vascular plants and may be essential in chloroplast protein processing. Treating tobacco seedlings with the peptide deformylase inhibitor actinonin resulted in leaf chlorosis and reduced growth and development, indicative of a systemic movement of the inhibitor. Photosystem II (PSII) activity was reduced, manifested as a significant decrease in the maximum quantum efficiency of photosystem II. Accumulation and assembly of nascent D1 protein into PSII monomers was also reduced, eventually leading to PSII disassembly and leaf necrosis. Processing and assembly of D1 protein in tobacco was a major and potentially critical target of peptide deformylase inhibition. These results confirm that N-terminal deformylation is an essential step in the accumulation and assembly of PSII subunit polypeptides in the chloroplasts of vascular plants.
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http://dx.doi.org/10.3732/ajb.91.9.1304DOI Listing
September 2004

A physical, enzymatic, and genetic characterization of perturbations in the seeds of the brownseed tomato mutants.

J Exp Bot 2004 May 8;55(399):961-73. Epub 2004 Apr 8.

Department of Horticulture, Plant Science Building, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0312, USA.

The brownseed mutants (bs(1), bs(2), and bs(4)) of tomato all possess dark testae and deleteriously affect seed germination speed and/or final percentage. Poor germination performance of the bs(1) but not the bs(4) mutant, was due to greater impediment to radicle egress. Testa toughening (bs(1)) was prevented by drying in N(2). However, poor germination speed was hardly affected by drying. GA(4+7) did not ameliorate germination percentage or speed (bs(1), bs(2)), whereas bs(4) seeds commenced radicle protrusion sooner and had a greater germination percentage. bs(1) mutant seeds have two times more catalase activity while those of bs(4) contained six times more peroxidase and almost two times more catalase activity than WTs. bs(4) release only half of the reactive oxygen species into the media than WT during imbibition. EPR detected the presence of free radicals in bs(1) and its WT. bs mutants were epistatic to 12 anthocyaninless mutations, at least some of which produce seeds of lighter than usual testa colour. Macro-arrays of subtractive, suppressive PCR products identified differentially regulated transcripts between seeds of bs(4) and WT. EST identity suggests bs(4) does not exit the developmental programme upon attaining maturity.
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http://dx.doi.org/10.1093/jxb/erh112DOI Listing
May 2004

Communication between the maternal testa and the embryo and/or endosperm affect testa attributes in tomato.

Plant Physiol 2003 Sep;133(1):145-60

Department of Horticulture, University of Kentucky, Lexington, KY 40546, USA.

Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 microM gibberellin(4 + 7) were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin(4 + 7) did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N(2). The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC196591PMC
http://dx.doi.org/10.1104/pp.103.022632DOI Listing
September 2003

Structure and catalytic mechanism of a SET domain protein methyltransferase.

Cell 2002 Oct;111(1):91-103

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Protein lysine methylation by SET domain enzymes regulates chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. The 2.6 A resolution structure of Rubisco large subunit methyltransferase in a pseudo-bisubstrate complex with S-adenosylhomocysteine and a HEPES ion reveals an all-beta architecture for the SET domain embedded within a larger alpha-helical enzyme fold. Conserved regions of the SET domain bind S-adenosylmethionine and substrate lysine at two sites connected by a pore. We propose that methyl transfer is catalyzed by a conserved Tyr at a narrow pore connecting the sites. The cofactor enters by a "back door" on the opposite side of the enzyme from substrate, promoting highly specific protein recognition and allowing addition of multiple methyl groups.
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http://dx.doi.org/10.1016/s0092-8674(02)01000-0DOI Listing
October 2002

Specificity of chloroplast-localized peptide deformylases as determined with peptide analogs of chloroplast-translated proteins.

Arch Biochem Biophys 2002 Oct;406(1):135-41

Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, N-32-D Agricultural Science Center North, University of Kentucky, Lexington, KY 40546-0091, USA.

Peptide deformylase (DEF; EC 3.5.1.88) removes the N-formyl group from nascent polypeptides. Two nuclear-encoded DEFs in Arabidopsis thaliana (At) are localized to chloroplasts, and thus, the N-termini of chloroplast-translated proteins may be a consequence of AtDEFs' substrate specificity. Using peptide analogs of select chloroplast-translated proteins, AtDEF1 activity was as much as 100-fold lower than AtDEF2 activity and showed little variance with peptide sequence. However, AtDEF2 activity was significantly influenced by peptide sequence, with the most efficiently processed substrate mimicking the N-terminus of the nascent D1 polypeptide, a core protein of photosystem II. Though AtDEF2's specificity was predictive of N-formyl retention for some chloroplast proteins, exceptions suggests that additional factors in vivo aid in determining the retention of an N-formyl group.
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http://dx.doi.org/10.1016/s0003-9861(02)00426-5DOI Listing
October 2002