Structure and regulatory mechanisms of inosine monophosphate-specific 5′-nucleotidase 1 = Inosine monophosphate-specific 5′-nucleotidase 1 의 효소 구조와 활성 조절 메커니즘 분석

국문 초록 (Abstract)

핵산 대사는 모든 생물체에서 세포 조절에 필수적인 역할을 한다. 퓨린 대사에서 이노신 일인산은 원핵생물에서 진핵생물에 이르기까지 중요한 분기점 역할을 한다. 흥미롭게도, 사카로미세스 세레비시아는 전형적인 퓨린 대사의 5'-핵산분해효소인 cytosolic 5'-nucleotiase Ⅱ (cN-Ⅱ) 를 결여하고 있으며, 대신 새롭게 발견된 5'-핵산분해효소 클래스인 IMP-specific 5'-nucleotiase 1 (ISN1) 에만 의존한다. cN-Ⅱ가 폭넓은 기질 특이성을 갖는 반면, 사카로미세스 세레비시아의 ISN1 (ScISN1)은 이노신 일인산에 대해 엄격한 기질 특이성을 보이며, 이를 퓨린 이화작용의 주요 중간체인 이노신으로 탈인산화한다. 본 학위 논문에서는 ScISN1의 결정구조, 생화학적 특성, 조절 메커니즘을 조사하여 이노신과 아데노신 삼인산에 의한 억제를 규명하였다. 구조 분석을 통해 이노신 일인산 결합이 국소적 영역의 구조 안정화와 서브 유닛 간 상호작용을 통한 전역적 구조 변화를 유도함을 확인했다. 생화학적 연구 결과, ScISN11은 아데노신 일인산-활성화 상동 효소인 말라리아 원충 유래 ISN1 (PfISN1)과는 대조적으로 기질에만 의존적인 구조적 변화를 나타냈다. PfISN1과의 서열 및 구조 비교를 통해 N-말단 및 C-말단 조절 요소에서의 주요 차이를 확인하였으며, 이는 기질 친화도와 효소 조절 메커니즘의 차이를 초래하는 것으로 제안할 수 있다. 이 연구는 ScISN1의 독특한 조절 메커니즘과 퓨린 대사에서 이노신 일인산 및 이노신 항상성 유지에서의 중요한 역할을 강조한다.

다국어 초록 (Multilingual Abstract)

Nucleotide metabolism is essential for cellular regulation across biological systems. In the purine metabolism, inosine monophosphate (IMP) serves as a critical junction point, connecting biosynthetic and catabolic pathways in organisms from prokaryotes to eukaryotes. Typically, the cytosolic 5′-nucleotidase Ⅱ (cN-Ⅱ), a ubiquitous enzyme across many species, dephosphorylates IMP into inosine which is a major intermediate in purine catabolism. Interestingly, Saccharomyces cerevisiae lacks the conventional enzyme cN-II and instead relies exclusively on IMP-specific 5′-nucleotidase 1 (ISN1), a newly identified class of 5′-nucleotidase. Unlike cN-Ⅱ with broad substrate specificity, ISN1 in S. cerevisiae (ScISN1) exhibits strict substrate specificity for IMP. This study investigated the structure, biochemical properties, and regulatory mechanisms of ScISN1 and revealed that inosine and adenosine triphosphate (ATP) are negatively regulate ScISN1 activity. Structural analyses demonstrated IMP-induced conformational changes, including disorder-to-order transitions and propagation through intersubunit interactions. Kinetic studies support IMP-dependent conformational changes in ScISN1 distinct from its homolog, ISN1 from Plasmodium falciparum (PfISN1), which is activated by ATP. Sequence and structural comparisons with PfISN1 further highlighted divergences of ScISN1 in N- and C-terminal elements, underlying differences in enzyme regulation. These findings highlight unique regulatory mechanisms of ScISN1 and its critical role in maintaining IMP and inosine homeostasis in purine metabolism.

다국어 초록 (Multilingual Abstract)

Nucleotide metabolism is essential for cellular regulation across biological systems. In the purine metabolism, inosine monophosphate (IMP) serves as a critical junction point, connecting biosynthetic and catabolic pathways in organisms from prokaryotes to eukaryotes. Typically, the cytosolic 5′-nucleotidase Ⅱ (cN-Ⅱ), a ubiquitous enzyme across many species, dephosphorylates IMP into inosine which is a major intermediate in purine catabolism. Interestingly, Saccharomyces cerevisiae lacks the conventional enzyme cN-II and instead relies exclusively on IMP-specific 5′-nucleotidase 1 (ISN1), a newly identified class of 5′-nucleotidase. Unlike cN-Ⅱ with broad substrate specificity, ISN1 in S. cerevisiae (ScISN1) exhibits strict substrate specificity for IMP. This study investigated the structure, biochemical properties, and regulatory mechanisms of ScISN1 and revealed that inosine and adenosine triphosphate (ATP) are negatively regulate ScISN1 activity. Structural analyses demonstrated IMP-induced conformational changes, including disorder-to-order transitions and propagation through intersubunit interactions. Kinetic studies support IMP-dependent conformational changes in ScISN1 distinct from its homolog, ISN1 from Plasmodium falciparum (PfISN1), which is activated by ATP. Sequence and structural comparisons with PfISN1 further highlighted divergences of ScISN1 in N- and C-terminal elements, underlying differences in enzyme regulation. These findings highlight unique regulatory mechanisms of ScISN1 and its critical role in maintaining IMP and inosine homeostasis in purine metabolism.

목차 (Table of Contents)

• Abstract ⅰ
• Table of Contents ⅱ
• List of Figures ⅳ
• List of Tables ⅷ
• List of Abbreviations ⅸ
• 1.1 Inroduction 1
• 1.1.1 Purine metabolism 1
• 1.1.2 5'-Nucleotidase and its physiological role in purine metabolism 13
• 1.1.3 Regulation of purine metabolism by a novel 5'-nucleotidase from Saccharomyces cerevisiae 23
• 1.1.4 Biochemical properties of PfISN1 29
• 1.1.5 Structure and allosteric regulation of PfISN1 31
• 1.2 Purpose of this study 43
• 2.1 Materials and methods 44
• 2.1.1 Cloning and purification of ScISN1 44
• 2.1.2 Crystallization and structure determination 53
• 2.1.3 Enzyme-coupled assay for phosphate 63
• 2.1.4 Analysis of kinetic data 75
• 2.1.5 Inorganic phosphate determination 76
• 2.1.6 HPLC 79
• 2.1.7 LC/MS 81
• 2.1.8 Nuclear magnetic resonance (NMR) measurements 82
• 2.1.9 Circular dichroism (CD) spectra 83
• 2.2 Results 84
• 2.2.1 Structure of inosine liganded ScISN1* 84
• 2.2.2 Structural features of the ScISN1*(D172A)-IMP complex 103
• 2.2.3 IMP-induced changes and intersubunit communication 122
• 2.2.4 Biochemical and kinetic analysis of ScISN1 128
• 2.2.5 Site-directed mutagenesis analysis of ScISN1 136
• 2.2.6 Regulatory effect of ATP and inosine on ScISN1 activity 152
• Discussion 168
• Conclusion 179
• References 180
• Accssion numbers 187
• Abstract in Korean 188