Elucidating beta-lactamase functional mechanisms via evolutionary conservation

通过进化保守阐明β-内酰胺酶的功能机制

• 批准号: 8432993
• 负责人: Donald JACOBS
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF NORTH CAROLINA CHARLOTTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432993
• 关键词: Active Sites; Allosteric Site; Amino Acid Sequence; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Benign; Binding; Bioinformatics; Biophysics; Cell Wall; Cephalosporin Resistance; Cephalosporins; Comparative Study; Conceptions; Data; Development; Entropy; Enzymes; Equilibrium; Evolution; Family; Financial compensation; Fright; Future; Gatekeeping; Generations; Genes; Gram-Negative Bacteria; Gram-Positive Bacterial Infections; Hydrolysis; Ions; Lactamase; Lactams; Lead; Mechanics; Mediating; Metals; Methods; Metric; Modeling; Mutation; Orthologous Gene; Pattern; Penetration; Penicillin Resistance; Penicillins; Peptide Sequence Determination; Phylogenetic Analysis; Phylogeny; Property; Proteins; Public Health; Resistance; Series; Serine; Structure; Structure-Activity Relationship; System; Thermodynamics; Variant; Work; Zinc; bacterial resistance; base; beta-Lactamase; combat; comparative; design; effective therapy; enthalpy; flexibility; global health; improved; insight; interest; member; molecular dynamics; novel therapeutics; protein structure; public health relevance; resistance mechanism

DESCRIPTION (provided by applicant): Penicillins and chemically related molecules are our most abundant and common used class of antibiotics, which are characterized by a conserved 4-atom ¿-lactam ring. Historically, they have been an effective treatment to gram-positive bacterial infections; however, the cell wall of gram-negative bacteria poses an effective barrier to antibiotic penetration. Conversely, second generation cephalosporins are also effective against gram-negative bacteria because they are able to penetrate the cell wall. Nevertheless, an increasing number of bacteria are resistant due to the enzyme ?-lactamase (BL). BL, which is expressed in the cell wall, hydrolyzes the ? -lactam ring, thus rendering the antibiotic ineffective. Due to decades of antibiotic overuse, BL enzymes have alarmingly evolved additional resistances that are now breaking down our last lines of defense. For example, extended spectrum ? -lactamases (ESBL) also hydrolyze the ? -lactam ring of cephalosporins, which have generally been resistant to BL activity. As such, a better understanding of BL resistance mechanisms is imperative so that new and more effective antibiotics can be developed quickly. There are four common classes of BL enzymes, which reflect specific sequence, structure and antibiotic resistance patterns. The Class A, C and D enzymes share a serine-based hydrolysis, whereas the catalytic mechanism of Class B enzymes is based on a zinc ion. However, little is known about how dynamics and stability vary across the superfamily. Are stability and/or dynamical mechanisms conserved across the superfamily? Are certain mechanisms critical to function? Can mechanistic differences help explain antibiotic resistance patterns? Is allostery conserved across the superfamily? These are the types of unanswered questions this proposal seeks to answer. To that end, we will employ a powerful and fast computational distance constraint model (DCM) to characterize the serine-based classes. While broad characterization across the BL superfamily has not yet been done, a small number of Class A structures have been studied by NMR and molecular dynamics simulation. Interestingly, these structures appear extraordinarily rigid, punctuated by flexible loop regions that may or may not be related to function. Our preliminary DCM characterizations across Class A enzymes reproduce these results. Even so, there is significant diversity within dynamical quantities across the family, which reflects evolutionary out-groups and, in many cases, parallels the onset of extended-spectrum activities. Taken together, these preliminary results highlight how the synthesis of biophysical descriptions with the paradigm of comparative bioinformatics synergistically improves the importance and accuracy of our characterizations. As such, we propose a series of additional studies along these lines to expand our understanding of BL structure and function, potentially paving the way to new therapeutic opportunities.

描述（由适用提供）：青霉素和化学相关的分子是我们最丰富，最常用的抗生素类别，其特征是保守的4 -ATOM� -lactam环。从历史上看，它们一直是革兰氏阳性细菌感染的有效治疗方法。但是，革兰氏阴性细菌的细胞壁具有有效的抗生素渗透障碍。相反，第二代头孢菌素也针对革兰氏阴性细菌有效，因为它们能够穿透细胞壁。然而，由于酶？-lactamase（BL），越来越多的细菌具有抗性。 BL，在细胞壁上表达的，水解了？ -lactam环，因此使抗生素无效。由于数十年的抗生素过度使用，BL酶令人震惊地发展了额外的抗药性，这正在打破我们的最后防御线。例如，扩展频谱？ - 头孢菌素的lactam环，通常对BL活性具有抗性。因此，必须更好地了解BL抗性机制，因此可以快速开发新的，更有效的抗生素。有四种常见的BL酶，它们反映了特定的序列，结构和抗生素抗性模式。 A类A，C和D酶具有基于丝氨酸的水解，而B类酶的催化机理基于锌离子。但是，对于整个超家族的动态和稳定性如何变化，知之甚少。在整个超家族中保存稳定性和/或动态机制吗？某些机制对功能至关重要吗？机械差异可以帮助解释抗生素抗性模式吗？在超家族中保存了变构吗？这些是该提案寻求回答的未解决问题的类型。为此，我们将采用强大而快速的计算距离约束模型（DCM）来表征基于丝氨酸的类。尽管尚未完成整个BL超家族的广泛表征，但NMR和分子动力学模拟已经研究了少数A类结构。有趣的是，这些结构看起来非常僵化，并由可能与功能无关的柔性环区域打断。我们跨A类酶的初步DCM字符重现了这些结果。即使这样，在整个动态数量中仍有大量多样性 这个家庭反映了进化的群体外部，在许多情况下，该家庭与扩展光谱活动的发作相似。综上所述，这些初步结果强调了生物物理描述与比较生物信息学范式的合成如何协同提高我们角色的重要性和准确性。因此，我们提出了一系列沿这些线路的其他研究，以扩大我们对BL结构和功能的理解，并有可能为新的治疗机会铺平道路。