Background: Lysine carbamylation facilitates metal coordination for enzymatic activities. Results: Structures of dihydropyrimidinase as the apo-and holoenzyme with one and two metals and its substrate/product complexes are determined.
Conclusion:The structures reveal four steps in the assembly of the holoprotein with the carbamylated lysine and two metal ions. Significance: The results illustrate how proteins exploit lysines and metals to accomplish lysine carbamylation and enzymatic functions.
Bacterial hydantoinase possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. How the carboxylated lysine and metal binding affect the activity of hydantoinase was investigated. A significant amount of iron was always found in Agrobacterium radiobacter hydantoinase purified from unsupplemented cobalt-, manganese-, or zinc-amended Escherichia coli cell cultures. A titration curve for the reactivation of apohydantoinase with cobalt indicates that the first metal was preferentially bound but did not give any enzyme activity until the second metal was also attached to the hydantoinase. The pH profiles of the metal-reconstituted hydantoinase were dependent on the specific metal ion bound to the active site, indicating a direct involvement of metal in catalysis. Mutation of the metal binding site residues, H57A, H59A, K148A, H181A, H237A, and D313A, completely abolished hydantoinase activity but preserved about half of the metal content, except for K148A, which lost both metals in its active site. However, the activity of K148A could be chemically rescued by short-chain carboxylic acids in the presence of cobalt, indicating that the carboxylated lysine was needed to coordinate the binuclear ion within the active site of hydantoinase. The mutant D313E enzyme was also active but resulted in a pH profile different from that of wild-type hydantoinase. A mechanism for hydantoinase involving metal, carboxylated K148, and D313 was proposed.
Based on the multiple sequence/structure analysis and with sufficient information from other members of the same enzyme families, the origin and mechanism of specific enzyme actions and proteins assembly can be clarified and predicted.
Protein tyrosine sulfation is a key post‐translational modification that mediates various critical functions, such as HIV entry, inflammation, coagulation, and sterility. Tyrosine O‐sulfation is catalyzed by membrane‐associated tyrosylprotein sulfotransferase (TPST, EC 2.8.2.20), which is responsible for the sulfation of secreted and transmembrane proteins. The sulfated proteins are generally attained from tissue extraction, chemical synthesis, and enzymatic catalysis. Enzymatic catalysis provides direct, specific and easy method for the identification and preparation of sulfated proteins. Currently, TPST is mainly acquired from nature tissues or cell cultures. In this report, the constant source of active and homogeneous TPST at large quantity was achieved through prokaryotic expression. Furthermore, a continuous and cheap 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) regenerating system was utilized to provide the activated sulfate for protein sulfation. This methodology rendered the increase in catalytic efficiency of TPST up to 10–100 folds than previous studies. Moreover, we applied this platform in vivo to produce target protein concurrently with sulfation on the specific tyrosine residue throughout bacterial cultivation. This platform will be beneficial to synthesize sulfated proteins and investigate the functions of protein sulfation. support: 98‐2627‐B‐009‐009
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