Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans

Abstract

Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.

High-Speed Atomic Force Microscopy Visualization of the Dynamics of the Multienzyme Fatty Acid Synthase

“…However, on the basis of the extent of its flexible linker, the mobility of the phosphopantetheinylated ACP in the static model is sufficient to reach a complete set of active sites within one reaction cleft at distances between active sites of up to 85 Å, while the opposite site reaction cleft remains out of reach (Figure ). However, in vitro mutant complementation and cross-linking assays indicated the interaction of one ACP with active sites of the condensing region from both reaction clefts. , Indeed, electron microscopy (EM) analysis of metazoan FAS demonstrated large-scale conformational variability with some correlation to functional states. , A similar degree of structural variability has been observed in other carrier protein-dependent multienzymes and suggests a general role of large-scale domain movements. , …”

“…19,20 A similar degree of structural variability has been observed in other carrier protein-dependent multienzymes and suggests a general role of large-scale domain movements. 21,22 Here, we report an experimental approach based on highspeed atomic force microscopy (HS-AFM) for studying the dynamics of individual multienzymes in aqueous solution approaching a temporal and spatial resolution required to follow large-scale conformational changes during catalysis. HS-AFM has been used to visualize dynamic protein systems such as myosin V on actin, 23 the F 1 -ATPase, 24 GroEL−GroES interaction, 25 and the nuclear pore complex.…”