The functional principle of eukaryotic molybdenum insertases

Abstract

The molybdenum cofactor (Moco) is a redox-active prosthetic group found in the active site of Moco-dependent enzymes, which are vitally important for life. Moco biosynthesis involves several enzymes that catalyze the subsequent conversion of GTP into cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), adenylated MPT (MPT-AMP), and finally Moco. While the underlying principles of cPMP, MPT, and MPT-AMP formation are well understood, the molybdenum insertase (Mo-insertase)-catalyzed final Moco maturation step is not. In the present study, we analyzed high-resolution X-ray datasets of the plant Mo-insertase Cnx1E that revealed two molybdate-binding sites within the active site, hence improving the current view on Cnx1E functionality. The presence of molybdate anions in either of these sites is tied to a distinctive backbone conformation, which we suggest to be essential for Mo-insertase molybdate selectivity and insertion efficiency.

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

“…Within the Cnx1E active site, MPT-AMP adopts a conformation that is different from the one found in the Cnx1G MPT-AMP co-structure [11] and that properly orients the dithiolene motif toward enzyme bound molybdate [14,15]. Subsequently the MPT-AMP phosphoric anhydride bond is hydrolyzed, a reaction that is believed to be the prerequisite for enzyme catalyzed molybdate insertion into the MPT dithiolene motif [13][14][15]. Recent work suggested that the Cnx1E catalyzed molybdate insertion reaction involves the relocation of molybdate from the initial oxo-anion binding site to the insertion site [15].…”

“…In vivo interaction studies revealed the plant MPT synthase complex and the molybdenum insertase (Mo-insertase) to interact with each other, thus providing the framework necessary for efficient, protected and directed metabolite transfer [6]. The molybdate insertion reaction involves both functional domains of Mo-insertases, namely Eand G-domain [7] whose role for the Mo-insertion reaction has been studied in detail using the plant (Arabidopsis thaliana) Mo-insertase Cnx1 as model enzyme [8][9][10][11][12][13][14][15]. Notably, these domains are reactive as separately expressed domains (prokaryotes) or fused together (eukaryotes, except the lower alga Chlamydomonas reinhardtii [16]).…”

“…Initially, the Mo-insertase G-domain catalyzes the ligation of an AMP molecule to the terminal phosphate group of MPT, yielding adenylated MPT (MPT-AMP, [11,12]). Subsequently, MPT-AMP is transferred to the Mo-insertase E-domain, which requires molybdate to be bound to the E-domain oxo-anion entry site [15]. Within the Cnx1E active site, MPT-AMP adopts a conformation that is different from the one found in the Cnx1G MPT-AMP co-structure [11] and that properly orients the dithiolene motif toward enzyme bound molybdate [14,15].…”

“…Subsequently, MPT-AMP is transferred to the Mo-insertase E-domain, which requires molybdate to be bound to the E-domain oxo-anion entry site [15]. Within the Cnx1E active site, MPT-AMP adopts a conformation that is different from the one found in the Cnx1G MPT-AMP co-structure [11] and that properly orients the dithiolene motif toward enzyme bound molybdate [14,15]. Subsequently the MPT-AMP phosphoric anhydride bond is hydrolyzed, a reaction that is believed to be the prerequisite for enzyme catalyzed molybdate insertion into the MPT dithiolene motif [13][14][15].…”

“…Subsequently the MPT-AMP phosphoric anhydride bond is hydrolyzed, a reaction that is believed to be the prerequisite for enzyme catalyzed molybdate insertion into the MPT dithiolene motif [13][14][15]. Recent work suggested that the Cnx1E catalyzed molybdate insertion reaction involves the relocation of molybdate from the initial oxo-anion binding site to the insertion site [15]. Upon synthesis Moco is transferred to the cellular user enzymes and/or to the cellular Moco transfer/storage system [2,6,17,18].…”