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Structure-Based Mechanistic Insights into ColB1, a Flavoprotein Functioning in-trans in the 2,2'-Bipyridine Assembly
Xueyang Ma1, Zhijun Tang2, Wenping Ding2
1State Key Laboratory of Natural and Biomimetic Drugs, Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China.
Abstract:
The recruitment of trans-acting enzymes by nonribosomal peptide synthetase (NRPS) assembly line is rarely reported. ColB1 is a flavin-dependent dehydrogenase that is recruited by an NRPS terminal condensation domain (Ct domain) and catalyzes peptidyl carrier protein (PCP)-tethered cysteine dehydrogenation in collismycin biosynthesis. We here report the crystal structure of ColB1 complexed with FAD and reveal a typical structural fold of acyl-CoA dehydrogenases (ACADs). However, ColB1 shows distinct structural features from ACADs in substrate recognition both at the entrance of and inside the active site. Site-directed mutagenesis and substrate modeling establish a Glu393-mediated catalytic mechanism, by which the cysteine substrate is sandwiched between Glu393 and FAD to facilitate Cα proton abstraction and Cβ hydride migration. A ColB1-PCP-Ct complex model is generated, providing structural basis for the unique recruitment interactions between ColB1 and the associated NRPS. These results add insights into the mechanisms by which trans-acting enzymes function in an assembly line.
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