Search

Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate‐dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate‐recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5–1.9 Å resolution using synchrotron X‐rays and an X‐ray free‐electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate‐binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate‐binding and reaction process of cyanase. [ABSTRACT FROM AUTHOR]

MalE is a maltose/maltodextrin‐binding protein (MBP) that plays a critical role in most bacterial maltose/maltodextrin‐transport systems. Previously reported wild‐type MBPs are monomers comprising an N‐terminal domain (NTD) and a C‐terminal domain (CTD), and maltose‐like molecules are recognized between the NTD and CTD and transported to the cell system. Because MBP does not undergo artificial dimerization, it is widely used as a tag for protein expression and purification. Here, the crystal structure of a domain‐swapped dimeric MalE from Salmonella enterica (named SeMalE) in complex with maltopentaose is reported for the first time, and its structure is distinct from typical monomeric MalE family members. In the domain‐swapped dimer, SeMalE comprises two subdomains: the NTD and CTD. The NTD and CTD of one molecule of SeMalE interact with the CTD and NTD of the partner molecule, respectively. The domain‐swapped dimeric conformation was stabilized by interactions between the NTDs, CTDs and linkers from two SeMalE molecules. Additionally, a maltopentaose molecule was found to be located at the interface between the NTD and CTD of different SeMalE molecules. These results provide new insights that will improve the understanding of maltodextrin‐binding MalE proteins. [ABSTRACT FROM AUTHOR]

The brain‐type fatty acid‐binding protein FABP7, which is expressed in astrocytes and neural progenitors, is a member of the intracellular lipid‐binding protein family. This protein is not only involved in various cellular functions such as metabolism, inflammation and energy homeostasis, but also in diseases such as cognitive disorders and tumors. Structures of unsaturated fatty acids, such as oleic acid (OA) and docosahexaenoic acid (DHA), bound to FABP7 have been elucidated; however, structures of saturated fatty acids bound to FABP7 remain unknown. To better understand fatty acid recognition, here the crystal structure of human brain‐type fatty acid‐binding protein FABP7 complexed with palmitic acid (PA), a saturated fatty acid, is reported at a resolution of 1.6 Å. The PA bound to the fatty acid‐binding pocket of FABP7 assumed a U‐shaped conformation. The carboxylate moiety of PA interacted with Tyr129, Arg127 and, via a water bridge, with Arg107 and Thr54, whereas its aliphatic chain was stabilized by hydrophobic interactions with Met21, Leu24, Thr30, Thr37, Pro39, Phe58 and Asp77. Structural comparison showed that PA, OA and DHA exhibited unique binding conformations in the fatty acid‐binding pocket, stabilized by distinct amino‐acid interactions. The binding of PA to FABP7 exhibits a unique binding conformation when compared with other human FABPs (FABP3–FABP5 and FABP8) expressed in other tissues. Based on the crystal and fatty acid structures, it was suggested that PA, which prefers a linear form in nature, required a greater conformational change in its aliphatic chain to bind to the fatty acid‐binding pocket in a U‐shaped conformation, compared with the cis configurations of OA or DHA. This, together with the length of the aliphatic chain, was considered to be one of the factors determining the binding affinity of PA to FABP7. These results provide a better understanding of fatty acid recognition by FABP7 and expand the knowledge of the binding of PA to FABPs. [ABSTRACT FROM AUTHOR]