通过对放线菌——北里孢菌（Kitasatospora sp.）CPCC 204717的基因组分析，鉴定出一个潜在编码吲哚二酮哌嗪天然产物生物合成基因簇（kcm），其结构包含环二肽合成酶（CDPS）、细胞色素P450酶（CYP450）、短链脱氢酶/还原酶（SDR）、调控因子及未知功能基因等模块。序列比对显示，该基因簇中P450编码基因与结核分枝杆菌（M. tuberculosis，Mtb） CYP121（Rv2276）具有53 %的序列同源性。基于此，本论文对这一基因簇功能进行了深入的探索。

首先，对次级代谢基因簇kcm进行异源表达，对其代谢产物进行分离与结构鉴定，从中发现了6个吲哚二酮哌嗪（Indole Diketopiperazine Alkaloid, IDKP）类化合物，其中化合物2、3是鸟嘌呤修饰型吲哚二酮哌嗪衍生物，化合物4、5具有独特的3-羟基-2-氧吲哚结构的吲哚二酮哌嗪类化合物。结合体内关键基因功能研究、体外酶促反应及分子系统发育分析等手段，上述化合物的编码基因KcmB被鉴定为一个特殊的具有三种催化功能的P450酶，可同时实现（1）催化吲哚二酮哌嗪(Cyclo (L-Trp-L-Tyr), cWW)的C3位与鸟嘌呤C8’’位发生C-C偶联；（2）介导cWW的N1位与鸟嘌呤C8’’位C-N偶联；（3）在cWW的吲哚环C2-C3位实现非经典连续加氧反应。通过对酶催化底物特异性考察，KcmB表现出一定的底物宽泛性。

在此基础上，本论文通过SPR、BLI、以及CYP121酶抑制实验，评价了KcmB编码产生产物对抗结核重要靶点CYP121 (Rv2276)的亲和力和抑制活性，发现KcmB催化产物能够与CYP121结合并发挥抑制作用。

总之，本论文对基因簇kcm进行异源表达和产物挖掘，得到了6个IDKP类化合物，其中化合物4、5为具有3-羟基-2-氧吲哚结构的新化合物，拓展了IDKP衍生物的结构多样性。同时对上述化合物的生物合成机制研究，发现了一个具有3种催化功能的P450蛋白KcmB，提出了对P450酶的新认识，为基于KcmB开发的新生物催化工具奠定科学基础。对化合物的CYP121抑制活性研究，发现了一类新的CYP121的抑制剂，为天然来源抗结核药物的研发提供参考。

Based on genomic analysis of Kitasatospora sp. CPCC 204717 ^{[9, 10]}, we identified a novel secondary metabolic gene cluster (kcm) comprising a cyclodipeptide synthase (CDPS), a cytochrome P450 enzyme (P450), a short-chain dehydrogenase/reductase (SDR), regulatory elements, and genes of unknown function. Sequence alignment revealed that the P450-encoding gene within this cluster shares 53 % sequence homology with Mycobacterium tuberculosis (Mtb) CYP121 (Rv2276). Through heterologous expression studies of the  kcm cluster—which includes a tRNA-dependent cyclodipeptide synthase (KcmA) and the P450 enzyme KcmB—we isolated and purified six indole diketopiperazine alkaloid (IDKP) compounds. Among these, compounds 2 and 3 are guanine-modified derivatives, while compounds 4 and 5 feature a unique 3-hydroxy-2-oxindole scaffold, indicating the cluster’s sophisticated catalytic capabilities. Integrated approaches, including gene knockout experiments, in vitro enzymatic assays, and molecular phylogenetic analysis, identified a rare multifunctional P450 enzyme, KcmB, capable of three distinct catalytic activities: 1. C–C coupling between the C3 position of compound 1 and the C8′′ position of guanine (generating 2); 2. C–N coupling between the N1 position of 1 and the C8′′ position of guanine (generating 3); 3 Non-classical sequential oxygenation at the C2–C3 positions of the indole ring (generating 4 and 5), as confirmed by ¹⁸O isotope labeling experiments, while preserving the intact indole structure.

Heterologous expression and substrate specificity assays further demonstrated that KcmB exhibits: 1. Broad compatibility with diverse redox coenzyme systems; 2. Substrate promiscuity toward nucleobases but strict specificity toward compound 3.

Given the high sequence homology between kcmB and the frontline antitubercular target CYP121 (Rv2276), this study innovatively proposes a reverse activity prediction strategy based on structural biology: KcmB-catalyzed products may target the active site of Mtb CYP121 to exert antitubercular effects. Using surface plasmon resonance (SPR), bio-layer interferometry (BLI), and LC-MS-based CYP121 inhibition assays, we validated the inhibitory effects of KcmB-derived products on CYP121, demonstrating the feasibility of this strategy. This breakthrough addresses two longstanding bottlenecks in natural product research—low efficiency in activity screening and unclear mechanisms of action.

In summary, heterologous expression and product mining of the kcm cluster yielded six IDKP derivatives, including novel compounds 4 and 5 with a 3-hydroxy-2-oxindole scaffold, significantly expanding IDKP structural diversity. The discovery of KcmB, a P450 enzyme with three catalytic functions, challenges the conventional paradigm of single-functionality in P450 enzymes. Furthermore, we propose a novel strategy leveraging structural similarity between homologous enzyme products to design small-molecule inhibitors that competitively occupy the CYP121 active cavity. This approach circumvents limitations of traditional substrate analogs, providing a new paradigm for developing pathogen-specific protein inhibitors and advancing efficient, sustainable drug synthesis technologies.