DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis

RNA上の遺伝情報を書き換える酵素であるDYWドメインの構造を解明 --植物オルガネラRNA編集のユニークな活性制御--. 京都大学プレスリリース. 2021-06-23.RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts—mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis

애기장대 유래 Pseudouridine Kinase의 구조와 생화학적 기능 분석

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2021.8. 이상기.RNA의 변형은 종류가 매우 다양하며 각각 RNA들의 안정성, mRNA와 단백질들간의 상호작용 그리고 단백질로의 번역과정에 효율성을 조절하는 등 다양한 역할을 담당한다. 대부분의 경우, RNA의 변형은 효소들에 의해서 특정 RNA에 부위 특이적으로 일어난다. 이들의 생물학적 역할, 생합성과정 관련 연구들과 비교해 변형된 RNA 의 분해 관련 연구는 비교적 미비하다. 가장 많이 발견되는 RNA 변형중 하나인 pseudouridine의 대사 관련 효소들이 최근 애기장대에서 동정이 되었다. 애기장대에서는 두개의 효소에 의해 pseudouridine의 분해를 촉매된다: PSEUDOURIDINE KINASE (PUKI) 와 PSEUDOURIDINE MONOPHOSPHATE GLYCOSYLASE (PUMY). PUKI와 PUMY는 pseudouridine을 pseudouridine monophosphate로 인산화 시키는 과정과 pseudouridine monophosphate를 uracil과 ribose 5’-phosphate로 가수분해하는 과정을 각각 촉매 한다. 차례로 해당 산물들은 pyrimidine의 일반적인 대사과정이나 재활용 과정에 사용된다. 본 학위논문에서는 pseudouridine 분해의 첫번째과정을 담당하는 효소인 애기장대 유래 PUKI의 단백질 삼차 구조를 분석하고, 규명된 단백질의 구조를 기반으로 기질 특이성에 관련된 인자들을 밝히고자 한다. AtPUKI는 PfkB family에 속하는 carbohydrate kinase로써, homodimer가 생물학적 기능 단위이다. 구조적인 특징으로는 α/β domain를 중심으로 가지고 있으며, 이로부터 유래한 β-strand domain로 구성이 되어있다. 흥미롭게도, β-strand domain는 dimerization interface를 제공하는 동시에 기질 특이성을 결정짓는 역할을 수행한다. AtPUKI, pseudouridine 그리고 ATP 결합구조를 토대로 AtPUKI에는 pseudouridine이 결합할 수 있는 독특한 결합부위가 존재하며, 이는 여러 개의 친수성 아미노산들에 의해 매개가 되는 것을 확인하였다. 특히, 인접한 단량체의 β-strand domain로부터 유래한 loop 또한 기질의 특이성을 결정짓는데 중요한 역할을 하며, 구조적인 변화의 수반이 요구된다는 것을 제시하였다. 이러한 동적인 특징은 AtPUKI가 uridine보다 pseudouridine에 높은 촉매 효율을 보이는 이유를 잘 설명한다. 두 기질들은 모두 AtPUKI와 비슷한 결합 친화도를 보이며 잘 결합하지만, 오직 pseudouridine만이 AtPUKI의 구조적인 변화를 야기하며 효소에 의해 효율적으로 분해된다(높은 turnover rate)는 것을 제시했다. 이러한 결과들은 어떻게 AtPUKI가 uridine을 포함하는 다른 pyrimidine nucleoside의 항상성을 방해하지 않고, pseudouridine의 인산화에만 관여하는지를 잘 설명하며, 더 나아가 PfkB family에 속하는 다양한 효소들의 구조적 그리고 기능적 다양성의 예시로서 사용될 수 있다.RNA modifications are chemically diverse site-specific events, achieved in most cases through an enzyme-dependent reaction. They regulate the stability of RNAs, mRNA–protein interactions, and translation efficiency. Compared to studies related to the biological role and biosynthesis process of RNA modifications, studies on the degradation of modified RNA are relatively incomplete. Recently, metabolic fate of pseudouridine, one of the most prevalent modified RNAs, was characterized in plant Arabidopsis thaliana. In the A. thaliana, two enzymes are responsible for pseudouridine degradation: PSEUDOURIDINE KINASE (PUKI) and PSEUDOURIDINE MONOPHOSPHATE GLYCOSYLASE (PUMY). PUKI and PUMY are involved in phosphorylating pseudouridine into pseudouridine monophosphate and hydrolyzing pseudouridine monophosphate into uracil and ribose 5’-phosphate, respectively. The resulting products can be subjected to a general pathway for pyrimidine catabolism or the salvage pathway. In this thesis, I conducted structural and biochemical analyses of PUKI from A. thaliana (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the phosphofructokinase B (PfkB) family of carbohydrate kinases, is a homodimeric α/β protein with a protruding small β-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, of which one is mediated by a loop from the small β-strand domain of the adjacent monomer. Conformational transition of the dimerized small β-strand domains containing active site residues is required for substrate specificity. This dynamic feature explains the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well to the AtPUKI with similar Km value, but only pseudouridine is turned over efficiently. These results provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis.Introduction 1 RNA modification 2 Chemical property and role of pseudouridine 5 Catabolism of canonical pyrimidine nucleoside 6 Catabolism of non-canonical nucleotides 10 Materials and Methods 17 Cloning and purification of AtPUKI 18 Crystallization and structure determination 27 Activity assays 33 Results and Discussion 37 Structure of unliganded AtPUKI 38 Structural homologs of AtPUKI and the structure of the monovalent cation-binding site 47 The ternary complex of AtPUKI with pseudouridine and ADP 55 Functional features of the AtPUKI active site residues 67 High fidelity of AtPUKI for pseudouridine 88 The substrate pocket of AtPUKI in comparison to ribokinase and adenosine kinase 90 The Mg2+-binding site and catalysis in AtPUKI 93 Reference 97 Accession numbers 102 Abstract in Korean 103박

Site directed mutagenesis as a tool to understand the catalytic mechanism of human cytidine deaminase.

Cytidine deaminase (CDA), is one of the enzymes involved in the pyrimidine salvage pathways, which catalyzes the formation of uridine and deoxyuridine by the hydrolytic deamination of cytidine and deoxycytidine, respectively. Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binds to each active site independently and the cooperativity between subunits has not been reported. CDA is able to recognize as substrates some antitumor and antiviral cytidine analogs rendering them pharmacologically inactive. In light of the role played by this enzyme, a deep knowledge of CDA active site and mechanism of catalysis is required. Site-directed mutagenesis, associated with molecular modeling studies, may be an important tool to discover the active site structure of an enzyme and consequently its mechanism of action. In this review are summarized the site-directed mutagenesis experiments performed on human CDA: through these studies it was possible to understand the role exerted by specific amino acid residues in CDA active site and in the contacts between subunits. The obtained results may open a way for designing new cytidine based drugs or more potent CDA inhibitors

Maize haplotype with a helitron-amplified cytidine deaminase gene copy

BACKGROUND: Genetic maps are based on recombination of orthologous gene sequences between different strains of the same species. Therefore, it was unexpected to find extensive non-collinearity of genes between different inbred strains of maize. Interestingly, disruption of gene collinearity can be caused among others by a rolling circle-type copy and paste mechanism facilitated by Helitrons. However, understanding the role of this type of gene amplification has been hampered by the lack of finding intact gene sequences within Helitrons. RESULTS: By aligning two haplotypes of the z1C1 locus of maize we found a Helitron that contains two genes, one encoding a putative cytidine deaminase and one a hypothetical protein with part of a 40S ribosomal protein. The cytidine deaminase gene, called ZmCDA3, has been copied from the ZmCDA1 gene on maize chromosome 7 about 4.5 million years ago (mya) after maize was formed by whole-genome duplication from two progenitors. Inbred lines contain gene copies of both progenitors, the ZmCDA1 and ZmCDA2 genes. Both genes diverged when the progenitors of maize split and are derived from the same progenitor as the rice OsCDA1 gene. The ZmCDA1 and ZmCDA2 genes are both transcribed in leaf and seed tissue, but transcripts of the paralogous ZmCDA3 gene have not been found yet. Based on their protein structure the maize CDA genes encode a nucleoside deaminase that is found in bacterial systems and is distinct from the mammalian RNA and/or DNA modifying enzymes. CONCLUSION: The conservation of a paralogous gene sequence encoding a cytidine deaminase gene over 4.5 million years suggests that Helitrons could add functional gene sequences to new chromosomal positions and thereby create new haplotypes. However, the function of such paralogous gene copies cannot be essential because they are not present in all maize strains. However, it is interesting to note that maize hybrids can outperform their inbred parents. Therefore, certain haplotypes may function only in combination with other haplotypes or under specialized environmental conditions

An overview of pentatricopeptide repeat proteins and their applications

AbstractPentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins which mediate several aspects of gene expression primarily in organelles but also in the nucleus. These proteins facilitate processing, splicing, editing, stability and translation of RNAs. While major advances in PPR research have been achieved with plant PPR proteins, the significance of non-plant PPR proteins is becoming of increasing importance. PPR proteins are classified into different subclasses based on their domain architecture, which is often a reflection of their function. This review provides an overview of the significant findings regarding the functions, evolution and applications of PPR proteins. Horizontal gene transfer appears to have played a major role in the sporadic phylogenetic distribution of different PPR subclasses in both eukaryotes and prokaryotes. Additionally, the use of synthetic biology and protein engineering to create designer PPR proteins to control gene expression in vivo is discussed. This review also highlights some of the aspects of PPR research that require more attention particularly in non-plant organisms. This includes the lack of research into the recently discovered PPR-TGM subclass, which is not only the first PPR subclass absent from plants but present in economically and clinically-relevant pathogens. Investigation into the structure and function of PPR-TGM proteins in these pathogens presents a novel opportunity for the exploitation of PPR proteins as drug targets to prevent disease

Structural basis for the substrate specificity and catalytic features of pseudouridine kinase from Arabidopsis thaliana

RNA modifications can regulate the stability of RNAs, mRNA-protein interactions, and translation efficiency. Pseudouridine is a prevalent RNA modification, and its metabolic fate after RNA turnover was recently characterized in eukaryotes, in the plant Arabidopsis thaliana. Here, we present structural and biochemical analyses of PSEUDOURIDINE KINASE from Arabidopsis (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the PfkB family of carbohydrate kinases, is a homodimeric α/β protein with a protruding small β-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, in particular at the nucleobase, by multiple hydrophilic interactions, of which one is mediated by a loop from the small β-strand domain of the adjacent monomer. Conformational transition of the dimerized small β-strand domains containing active site residues is required for substrate specificity. These dynamic features explain the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well (similar Km), but only pseudouridine is turned over efficiently. Our studies provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research

DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis

RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasi

Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9

In flowering plant plastids and mitochondria, multiple organellar RNA editing factor (MORF/RIP) proteins are required at most sites for efficient C to U RNA editing catalyzed by the RNA editosome. MORF proteins harbor a conserved stretch of residues (MORF-box), form homo- and heteromers and interact with selected PPR (pentatricopeptide repeat) proteins, which recognize each editing site. The molecular function of the MORF-box remains elusive since it shares no sequence similarity with known domains. We determined structures of the A. thaliana mitochondrial MORF1 and chloroplast MORF9 MORF-boxes which both adopt a novel globular fold (MORF domain). Our structures state a paradigmatic model for MORF domains and their specific dimerization via a hydrophobic interface. We cross-validate the interface by yeast two-hybrid studies and pulldown assays employing structure-based mutants. We find a structural similarity of the MORF domain to an N-terminal ferredoxin-like domain (NFLD), which confers RNA substrate positioning in bacterial 4-thio-uracil tRNA synthetases, implying direct RNA contacts of MORF proteins during RNA editing. With the MORF1 and MORF9 structures we elucidate a yet unknown fold, corroborate MORF interaction studies, validate the mechanism of MORF multimerization by structure-based mutants and pave the way towards a complete structural characterization of the plant RNA editosome