Mingers Toni, Barthels Stefan, Mass Violetta, Acuña José Manuel Borrero-de, Biedendieck Rebekka, Cooke Ana, Dailey Tamara A, Gerdes Svetlana, Blankenfeldt Wulf, Dailey Harry A, Warren Martin J, Jahn Martina, Jahn Dieter
Institute of Microbiology, University of Technical Engineering, Braunschweig, Germany.
Pieris Pharmaceuticals GmbH, Hallbergmoos, Germany.
Front Microbiol. 2024 Mar 13;15:1378989. doi: 10.3389/fmicb.2024.1378989. eCollection 2024.
Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in , employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called , encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from () . The recombinantly produced, purified, and monomeric YtpQ (CgoN) protein is shown to catalyze the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III. Minimal non-enzymatic conversion of coproporphyrinogen III was observed under the anaerobic test conditions employed in this study. FAD was identified as a cofactor, and menadione served as an artificial acceptor for the six abstracted electrons, with a value of 3.95 μmol/L and a of 0.63 per min for the substrate. The resulting coproporphyrin III, in turn, acts as an effective substrate for the subsequent enzyme of the pathway, the coproporphyrin III ferrochelatase (CpfC). Under aerobic conditions, oxygen directly serves as an electron acceptor, but is replaced by the more efficient action of menadione. An AlphaFold2 model of the enzyme suggests that YtpQ adopts a compact triangular shape consisting of three domains. The N-terminal domain appears to be flexible with respect to the rest of the structure, potentially creating a ligand binding site that opens and closes during the catalytic cycle. A catalytic mechanism similar to the oxygen-independent protoporphyrinogen IX oxidase PgoH1 (HemG), based on the flavin-dependent abstraction of six electrons from coproporphyrinogen III and their potential quinone-dependent transfer to a membrane-localized electron transport chain, is proposed.
自然界利用三种不同的途径来合成必需的酶辅因子血红素。以粪卟啉原III为依赖的途径主要存在于……中，该途径利用一种依赖氧气的粪卟啉原III氧化酶（CgoX），将粪卟啉原III转化为粪卟啉III。在本研究中，我们报告了基于生物信息学鉴定出一个名为……的基因，它编码一种假定的不依赖氧气的对应物，我们提议将其命名为CgoN，该基因来自……（……）。重组产生、纯化的单体YtpQ（CgoN）蛋白被证明能催化粪卟啉原III不依赖氧气转化为粪卟啉III。在本研究采用的厌氧测试条件下，观察到粪卟啉原III的非酶促转化极少。黄素腺嘌呤二核苷酸（FAD）被鉴定为一种辅因子，甲萘醌作为六个被提取电子的人工受体，底物的米氏常数（Km）值为3.95 μmol/L，每分钟的催化常数（kcat）为0.63。生成的粪卟啉III继而作为该途径后续酶——粪卟啉III亚铁螯合酶（CpfC）的有效底物。在有氧条件下，氧气直接作为电子受体，但被甲萘醌更有效的作用所取代。该酶的AlphaFold2模型表明，YtpQ呈紧凑的三角形，由三个结构域组成。N端结构域相对于结构的其余部分似乎较为灵活，可能形成一个在催化循环中打开和关闭的配体结合位点。基于从粪卟啉原III中黄素依赖地提取六个电子以及它们可能依赖醌转移到膜定位的电子传递链，提出了一种类似于不依赖氧气的原卟啉原IX氧化酶PgoH1（HemG）的催化机制。
