Predicting enzyme metalation to identify new therapeutic targets in infectious diseases

微生物金属酶防御宿主的应激反应并合成致病所需的营养物质。因此，微生物代谢特有的金属酶是治疗干预的有吸引力的目标，但抑制剂的发现需要了解生理金属辅助因子。关键的是，在试管中研究时，生物学上重要的金属并不总是最紧密地结合感兴趣的酶，这是欧文-威廉姆斯系列过渡金属配体稳定性的结果。这种明显的矛盾在细胞中通过缓冲不同水平的金属可用性来克服。我们最近开发了工具来确定这些缓冲水平，从而克服了识别各种金属蛋白的生理金属辅因子的挑战。难辨梭状芽胞杆菌是医院获得性腹泻的主要病因，其传播周期的重要组成部分是产孢。事实上，孢子是感染因子，负责感染和复发。孢子形成的全球调控是已知的，但在这一过程中对营养物质（包括金属）的需求和供应的研究很少。因此，产孢病原体提供了独特的金属酶治疗靶点，当被抑制时，将限制感染的传播，因为孢子减少。为了确定芽孢所必需的梭状芽孢杆菌酶的正确金属辅因子，金属有效性将首先使用我们最近开发的方法来确定。梭状芽胞杆菌金属调节蛋白将被过度表达和纯化，以便使用光谱方法确定金属和dna结合亲和力。这些亲和力值将用于确定细胞内生理上可用的金属水平。然后，将测定纯化的梭状芽孢杆菌酶的金属亲和力，以确定生理金属辅因子，即与计算出的可用性相匹配的辅因子。一项补充的研究将通过关注已知的含有一种或多种关键金属酶的生物合成途径来研究金属有效性和营养供应对梭状芽孢杆菌产孢效率的关系。

Microbial metalloenzymes defend against host stress responses and synthesize nutrients required for pathogenesis. Metalloenzymes unique to microbial metabolism are therefore attractive targets for therapeutic intervention, but inhibitor discovery requires knowledge of the physiological metal cofactor. Critically, the biologically important metal does not always bind the enzyme of interest most tightly when studied in the test tube, a consequence of the Irving-Williams series of transition metal-ligand stabilities. This apparent paradox is overcome in cells by buffering metal availability to differing levels. We have recently developed the tools to determine these buffered levels and thereby overcome the challenge of identifying the physiological metal cofactor of a variety of metalloproteins. Clostridium difficile, the leading cause of hospital-acquired diarrhoea, undergoes sporulation as an essential part of its transmission cycle. Indeed, spores are the infective agent, responsible for infection as well as recurrency. Global regulation of sporulation is known but the requirement for and supply of nutrients, including metals, during this process is poorly studied. Sporulating pathogens therefore offer unique metalloenzyme therapeutic targets that, when inhibited, would limit the spread of infection due to diminished sporulation. To determine the correct metal cofactors of Clostridium enzymes essential for sporulation, metal availabilities will first be determined using our recently developed approach. Clostridium metalloregulatory proteins will be overexpressed and purified to enable determination of metal and DNA-binding affinities using spectroscopic methods. These affinity values will be used to define physiologically available metal levels within the cell. Then, metal affinities of purified Clostridium enzymes will be determined to identify the physiological metal cofactor, ie, the one that matches calculated availability. A complementary line of investigation will examine the relationship between metal availability and nutrient supply on Clostridium sporulation efficiency by focusing on known biosynthetic pathways containing one or more key metalloenzymes.