Crystal structure of the catalytic core of Saccharomyces cerevesiae histone demethylase Rph1: insights into the substrate specificity and catalytic mechanism

2010 ◽  
Vol 433 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Yuanyuan Chang ◽  
Jian Wu ◽  
Xia-Jing Tong ◽  
Jin-Qiu Zhou ◽  
Jianping Ding
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Histone Demethylase ◽  
Structural Motif ◽  
Catalytic Core ◽  
Conserved Residues ◽  
Demethylase Activity ◽  
Jmjc Domain ◽  
Saccharomyces Cerevesiae ◽  
Binding Cleft

Saccharomyces cerevesiae Rph1 is a histone demethylase orthologous to human JMJD2A (Jumonji-domain-containing protein 2A) that can specifically demethylate tri- and di-methylated Lys36 of histone H3. c-Rph1, the catalytic core of Rph1, is responsible for the demethylase activity, which is essential for the transcription elongation of some actively transcribed genes. In the present work, we report the crystal structures of c-Rph1 in apo form and in complex with Ni2+ and α-KG [2-oxoglutarate (α-ketoglutarate)]. The structure of c-Rph1 is composed of a JmjN (Jumonji N) domain, a long β-hairpin, a mixed structural motif and a JmjC domain. The α-KG cofactor forms hydrogen-bonding interactions with the side chains of conserved residues, and the Ni2+ ion at the active site is chelated by conserved residues and the cofactor. Structural comparison of Rph1 with JMJD2A indicates that the substrate-binding cleft of Rph1 is formed with several structural elements of the JmjC domain, the long β-hairpin and the mixed structural motif; and the methylated Lys36 of H3 is recognized by several conserved residues of the JmjC domain. In vitro biochemical results show that mutations of the key residues at the catalytic centre and in the substrate-binding cleft abolish the demethylase activity. In vivo growth phenotype analyses also demonstrate that these residues are essential for its functional roles in transcription elongation. Taken together, our structural and biological data provide insights into the molecular basis of the histone demethylase activity and the substrate specificity of Rph1.

JmjN interacts with JmjC to ensure selective proteolysis of Gis1 by the proteasome

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2694-2701 ◽  
Author(s):  
Zhenzhen Quan ◽  
Stephen G. Oliver ◽  
Nianshu Zhang
Keyword(s):  
Structural Unit ◽  
Transcription Activation ◽  
Histone Demethylase ◽  
Transcription Activity ◽  
The Core ◽  
Demethylase Activity ◽  
Jmjc Domain ◽  
Β Sheets ◽  
The Stability ◽  
Selective Proteolysis

A group of JmjC domain-containing proteins also harbour JmjN domains. Although the JmjC domain is known to possess histone demethylase activity, the function of the JmjN domain remains largely undetermined. Previously, we have demonstrated that the yeast Gis1 transcription factor, bearing both JmjN and JmjC domains at its N terminus, is subject to proteasome-mediated selective proteolysis to downregulate its transcription activation ability. Here, we reveal that the JmjN and JmjC domains interact with each other through two β-sheets, one in each domain. Removal of either or both β-strands or the entire JmjN domain leads to complete degradation of Gis1, mediated partially by the proteasome. Mutating the core residues essential for histone demethylase activity demonstrated for other JmjC-containing proteins or deleting both Jumonji domains enhances the transcription activity of Gis1, but has no impact on its selective proteolysis by the proteasome. Together, these data suggest that JmjN and JmjC interact physically to form a structural unit that ensures the stability and appropriate transcription activity of Gis1.

scholarly journals Heme, A Metabolic Sensor, Directly Regulates the Activity of the KDM4 Histone Demethylase Family and Their Interactions with Partner Proteins

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 773
Author(s):  
Purna Chaitanya Konduri ◽  
Tianyuan Wang ◽  
Narges Salamat ◽  
Li Zhang
Keyword(s):  
Histone Demethylase ◽  
Interacting Proteins ◽  
Cellular Functions ◽  
Functional Activities ◽  
Nutritional Conditions ◽  
Demethylase Activity ◽  
Jmjc Domain ◽  
Metabolic Sensor ◽  
Heme Regulation ◽  
Partner Proteins

The KDM4 histone demethylase subfamily is constituted of yeast JmjC domain-containing proteins, such as Gis1, and human Gis1 orthologues, such as KDM4A/B/C. KDM4 proteins have important functions in regulating chromatin structure and gene expression in response to metabolic and nutritional stimuli. Heme acts as a versatile signaling molecule to regulate important cellular functions in diverse organisms ranging from bacteria to humans. Here, using purified KDM4 proteins containing the JmjN/C domain, we showed that heme stimulates the histone demethylase activity of the JmjN/C domains of KDM4A and Cas well as full-length Gis1. Furthermore, we found that the C-terminal regions of KDM4 proteins, like that of Gis1, can confer heme regulation when fused to an unrelated transcriptional activator. Interestingly, biochemical pull-down of Gis1-interacting proteins followed by mass spectrometry identified 147 unique proteins associated with Gis1 under heme-sufficient and/or heme-deficient conditions. These 147 proteins included a significant number of heterocyclic compound-binding proteins, Ubl-conjugated proteins, metabolic enzymes/proteins, and acetylated proteins. These results suggested that KDM4s interact with diverse cellular proteins to form a complex network to sense metabolic and nutritional conditions like heme levels and respond by altering their interactions with other proteins and functional activities, such as histone demethylation.

scholarly journals Noncatalytic Function of a JmjC Domain Protein Disrupts Heterochromatin

2019 ◽  
Vol 12 ◽  
pp. 251686571986224
Author(s):  
Kehan Bao ◽  
Songtao Jia
Keyword(s):  
Transcriptional Regulation ◽  
Cancer Cells ◽  
Histone Acetyltransferase ◽  
Histone Demethylase ◽  
Epigenetic Landscape ◽  
Disease Etiology ◽  
Chromatin Regulators ◽  
Demethylase Activity ◽  
Jmjc Domain ◽  
Domain Protein

Chromatin-modifying enzymes are frequently overexpressed in cancer cells, and their enzymatic activities play important roles in changing the epigenetic landscape responsible for tumorigenesis. However, many of these proteins also execute noncatalytic functions, which are poorly understood. In fission yeast, overexpression of Epe1, a histone demethylase homolog, causes heterochromatin defects. Interestingly, in our recent work, we discovered that overexpressed Epe1 recruits SAGA, a histone acetyltransferase complex important for transcriptional regulation, to disrupt heterochromatin, independent of its demethylase activity. Our findings suggest that overexpressed chromatin-modifying enzymes can alter the epigenetic landscape through changing their proteomic environments, an area that needs to be further explored in dissecting disease etiology associated with overexpression of chromatin regulators.

Structure of the JmjC-domain-containing protein JMJD5

2013 ◽  
Vol 69 (10) ◽  
pp. 1911-1920 ◽  
Author(s):  
Haipeng Wang ◽  
Xing Zhou ◽  
Minhao Wu ◽  
Chengliang Wang ◽  
Xiaoqin Zhang ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Embryonic Cell ◽  
Histone Demethylase ◽  
Amino Acid Residues ◽  
Lysine Methylation ◽  
Post Translational Modification ◽  
Active Centre ◽  
Histone Tails ◽  
Demethylase Activity ◽  
Jmjc Domain

The post-translational modification of histone tails is the principal process controlling epigenetic regulation in eukaryotes. The lysine methylation of histones is dynamically regulated by two distinct classes of enzymes: methyltransferases and demethylases. JMJD5, which plays an important role in cell-cycle progression, circadian rhythms and embryonic cell proliferation, has been shown to be a JmjC-domain-containing histone demethylase with enzymatic activity towards H3K36me2. Here, the crystal structure of human JMJD5 lacking the N-terminal 175 amino-acid residues is reported. The structure showed that the Gln275, Trp310 and Trp414 side chains might block the insertion of methylated lysine into the active centre of JMJD5, suppressing the histone demethylase activity of the truncated JMJD5 construct. A comparison of the structure of JMJD5 with that of FIH, a well characterized protein hydroxylase, revealed that human JMJD5 might function as a protein hydroxylase. The interaction between JMJD5 and the core histone octamer proteins indicated that the histone proteins could be potential substrates for JMJD5.

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme

Nature ◽  
1992 ◽  
Vol 358 (6382) ◽  
pp. 123-128 ◽  
Author(s):  
Anna Marie Pyle ◽  
Felicia L. Murphy ◽  
Thomas R. Cech
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
Catalytic Core ◽  
Substrate Binding Site

scholarly journals The activation loop and substrate-binding cleft of glutaminase C are allosterically coupled

2019 ◽  
Vol 295 (5) ◽  
pp. 1328-1337
Author(s):  
Yunxing Li ◽  
Sekar Ramachandran ◽  
Thuy-Tien T. Nguyen ◽  
Clint A. Stalnecker ◽  
Richard A. Cerione ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Fluorescence Emission ◽  
Reciprocal Relationship ◽  
Tryptophan Residue ◽  
Substrate Affinity ◽  
Activation Loop ◽  
Conformational Coupling ◽  
Binding Cleft ◽  
Glutaminase Activity

The glutaminase C (GAC) isoform of mitochondrial glutaminase is overexpressed in many cancer cells and therefore represents a potential therapeutic target. Understanding the regulation of GAC activity has been guided by the development of spectroscopic approaches that measure glutaminase activity in real time. Previously, we engineered a GAC protein (GAC(F327W)) in which a tryptophan residue is substituted for phenylalanine in an activation loop to explore the role of this loop in enzyme activity. We showed that the fluorescence emission of Trp-327 is enhanced in response to activator binding, but quenched by inhibitors of the BPTES class that bind to the GAC tetramer and contact the activation loop, thereby constraining it in an inactive conformation. In the present work, we took advantage of a tryptophan substitution at position 471, proximal to the GAC catalytic site, to examine the conformational coupling between the activation loop and the substrate-binding cleft, separated by ∼16 Å. Comparison of glutamine binding in the presence or absence of the BPTES analog CB-839 revealed a reciprocal relationship between the constraints imposed on the activation loop position and the affinity of GAC for substrate. Binding of the inhibitor weakened the affinity of GAC for glutamine, whereas activating anions such as Pi increased this affinity. These results indicate that the conformations of the activation loop and the substrate-binding cleft in GAC are allosterically coupled and that this coupling determines substrate affinity and enzymatic activity and explains the activities of CB-839, which is currently in clinical trials.

scholarly journals Characterization and Stress Response of the JmjC Domain-Containing Histone Demethylase Gene Family in the Allotetraploid Cotton Species Gossypium hirsutum

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1617
Author(s):  
Jie Zhang ◽  
Junping Feng ◽  
Wei Liu ◽  
Zhongying Ren ◽  
Junjie Zhao ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Stress Response ◽  
Gossypium Hirsutum ◽  
Gene Family ◽  
Function Analysis ◽  
Histone Demethylase ◽  
Abiotic Stress Response ◽  
Cotton Species ◽  
Jmjc Domain ◽  
Allotetraploid Cotton

Histone modification is an important epigenetic modification that controls gene transcriptional regulation in eukaryotes. Histone methylation is accomplished by histone methyltransferase and can occur on two amino acid residues, arginine and lysine. JumonjiC (JmjC) domain-containing histone demethylase regulates gene transcription and chromatin structure by changing the methylation state of the lysine residue site and plays an important role in plant growth and development. In this study, we carried out genome-wide identification and comprehensive analysis of JmjC genes in the allotetraploid cotton species Gossypium hirsutum. In total, 50 JmjC genes were identified and in G. hirsutum, and 25 JmjC genes were identified in its two diploid progenitors, G. arboreum and G. raimondii, respectively. Phylogenetic analysis divided these JmjC genes into five subfamilies. A collinearity analysis of the two subgenomes of G. hirsutum and the genomes of G. arboreum and G. raimondii uncovered a one-to-one relationship between homologous genes of the JmjC gene family. Most homologs in the JmjC gene family between A and D subgenomes of G. hirsutum have similar exon-intron structures, which indicated that JmjC family genes were conserved after the polyploidization. All G. hirsutumJmjC genes were found to have a typical JmjC domain, and some genes also possess other special domains important for their function. Analysis of promoter regions revealed that cis-acting elements, such as those related to hormone and abiotic stress response, were enriched in G. hirsutum JmjC genes. According to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, most G. hirsutumJmjC genes had high abundance expression at developmental stages of fibers, suggesting that they might participate in cotton fiber development. In addition, some G. hirsutumJmjC genes were found to have different degrees of response to cold or osmotic stress, thus indicating their potential role in these types of abiotic stress response. Our results provide useful information for understanding the evolutionary history and biological function of JmjC genes in cotton.

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