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

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).

Sulfur-Oxygen Chalcogen Bonding Mediates AdoMet Recognition in the Lysine Methyltransferase SET7/9.

Recent studies have demonstrated that carbon-oxygen (CH···O) hydrogen bonds have important roles in S-adenosylmethionine (AdoMet) recognition and catalysis in methyltransferases. Here, we investigate noncovalent interactions that occur between the AdoMet sulfur cation and oxygen atoms in methyltransferase active sites. These interactions represent sulfur-oxygen (S···O) chalcogen bonds in which the oxygen atom donates a lone pair of electrons to the σ antibonding orbital of the AdoMet sulfur atom.

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes.

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

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.

Calmodulin-mediated signal transduction pathways in Arabidopsis are fine-tuned by methylation.

Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds.

An intergenic region shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue specific and stress inducible bidirectional promoter analyzed in transgenic arabidopsis and tobacco plants.

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles.

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

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.

Human calmodulin methyltransferase: expression, activity on calmodulin, and Hsp90 dependence.

Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34) has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels.

Utilization of a calmodulin lysine methyltransferase co-expression system for the generation of a combinatorial library of post-translationally modified proteins.

By successfully incorporating sequence diversity into proteins, combinatorial libraries have been a staple technology used in protein engineering, directed evolution, and synthetic biology for generating proteins with novel specificities and activities. However, these approaches mostly overlook the incorporations of post-translational modifications, which nature extensively uses for modulating protein activities in vivo. As an initial step of incorporating post-translational modifications into combinatorial libraries, we present a bacterial co-expression system, utilizing a recently characterized calmodulin methyltransferase (CaM KMT), to trimethylate a combinatorial library of the calmodulin central linker region.

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

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.

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

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.

Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2.

Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether.

Rubisco in complex with Rubisco large subunit methyltransferase.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme.

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

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.

Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture.

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.

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

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.

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.

Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.

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

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.

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

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.

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

Transgenic tobacco plants expressing three different forms of Arabidopsis plant peptide deformylase (AtDEF1.1, AtDEF1.2 and AtDEF2; EC 3.

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

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.

Phylloplanins of tobacco are defensive proteins deployed on aerial surfaces by short glandular trichomes.

In plants, defensive proteins secreted to leaf aerial surfaces have not previously been considered to be a strategy of pathogen resistance, and the general occurrence of leaf surface proteins is not generally recognized. We found that leaf water washes (LWW) of the experimental plant Nicotiana tabacum tobacco introduction (TI) 1068 contained highly hydrophobic, basic proteins that inhibited spore germination and leaf infection by the oomycete pathogen Peronospora tabacina. We termed these surface-localized proteins tobacco phylloplanins, and we isolated the novel gene T-Phylloplanin (for Tobacco Phylloplanin) and its promoter from N.

Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT.

SET domain protein methyltransferases catalyze the transfer of methyl groups from the cofactor S-adenosylmethionine (AdoMet) to specific lysine residues of protein substrates, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex. The crystal structures of pea Rubisco large subunit methyltransferase (LSMT) in ternary complexes with either lysine or epsilon-N-methyllysine (MeLys) and the product S-adenosylhomocysteine (AdoHcy) were determined to resolutions of 2.65 and 2.

The life of ribulose 1,5-bisphosphate carboxylase/oxygenase--posttranslational facts and mysteries.

The life of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), from gene to protein to irreplaceable component of photosynthetic CO2 assimilation, has successfully served as a model for a number of essential cellular processes centered on protein chemistry and amino acid modifications. Once translated, the two subunits of Rubisco undergo a myriad of co- and posttranslational modifications accompanied by constant interactions with structurally modifying enzymes. Even after final assembly, the essential role played by Rubisco in photosynthetic CO2 assimilation is dependent on continuous conformation modifications by Rubisco activase.