Huang Wentao, Reinhardt Jakob K, Tian Anru, Zhang Xiao, Li Binghui, Gould Noah, Nallapati Sashirekha, Ivanov Alexander R, Wang Yi, Guo Jason J, Budil David E, Weng Jing-Ke
Institute for Plant-Human Interface, Northeastern University, Boston, MA, 02115, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Angew Chem Int Ed Engl. 2025 Sep 15;64(38):e202512449. doi: 10.1002/anie.202512449. Epub 2025 Aug 22.
DUF3328 is a protein family widely found in fungal natural product biosynthesis pathways. Although DUF3328 proteins have long been implicated in diverse modifications of inert C(sp)─H bonds, including halogenation, hydroxylation, and macrocyclization, the biochemical properties and catalytic mechanisms of DUF3328 proteins remain elusive. Here, we report the characterization of the DUF3328 protein CctR, which catalyzes C(sp)─H hydroxylation of fungal cyclic peptide cyclochlorotine. Through AlphaFold modeling, in vitro biochemical characterization, and spectroscopic analysis, we demonstrate that CctR is a membrane-associated copper-dependent enzyme that functions as a homodimer. The dimerization of CctR is mediated by its transmembrane helix, a four-helix coiled coil, and C-terminal disulfide bonds. The conserved HxxHC(x)HxxHC motif, characteristic of the DUF3328 superfamily, is anchored on the dimerization interface and forms a binuclear copper coordination center. Moreover, we show that CctR is dioxygen-dependent and requires electron input for the hydroxylation reaction. Together, these findings define DUF3328 as a previously unrecognized family of binuclear copper-dependent metalloenzymes, capable of catalyzing diverse chemical transformations, and lay the groundwork for future discovery of novel biocatalysts within this widespread enzyme class.
DUF3328是一个广泛存在于真菌天然产物生物合成途径中的蛋白质家族。尽管长期以来人们一直认为DUF3328蛋白参与了惰性C(sp)─H键的多种修饰，包括卤化、羟基化和大环化，但DUF3328蛋白的生化特性和催化机制仍然不清楚。在此，我们报道了DUF3328蛋白CctR的特性，它催化真菌环肽环氯肽素的C(sp)─H羟基化反应。通过AlphaFold建模、体外生化特性分析和光谱分析，我们证明CctR是一种与膜相关的铜依赖性酶，以同二聚体形式发挥作用。CctR的二聚化由其跨膜螺旋、四螺旋卷曲螺旋和C末端二硫键介导。DUF3328超家族特有的保守HxxHC(x)HxxHC基序锚定在二聚化界面上，形成一个双核铜配位中心。此外，我们表明CctR是双氧依赖性的，并且羟基化反应需要电子输入。这些发现共同将DUF3328定义为一个以前未被认识的双核铜依赖性金属酶家族，能够催化多种化学转化反应，并为今后在这一广泛的酶类中发现新型生物催化剂奠定了基础。
