Mechanism of molybdate insertion into pterin-based molybdenum cofactors

Abstract

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum (Mo) in Moco is the pyranopterin dithiolene (molybdopterin, MPT); however, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

The History of the Molybdenum Cofactor—A Personal View

“…For a decade, we did not touch Cnx1; then, my former Ph.D. student, Tobias Kruse, who meanwhile had set up his own team in my group, restarted Cnx1 research. His team determined the atomic structure of the E-domain [ 119 ] and revised our old model by showing that no activation of molybdate was necessary for insertion into MPT [ 120 , 121 ]. Details are given in Tobias Kruse’s review in this Special Issue.…”

Spectroscopic Studies of Mononuclear Molybdenum Enzyme Centers

“…XAS studies of the molybdenum insertase Cnx1 double variant S269D D274S has provided considerable insight into the mechanism of molybdenum cofactor (Moco) formation, with the insertion of Mo from molybdate representing the last step in Moco biosynthesis [ 97 , 98 ]. The Mo K-edge exhibits an intense pre-edge feature assignable as an “oxo-edge” transition whose importance lies in the fact that its intensity correlates with the number of oxo donor ligands bound to the Mo ion [ 99 , 100 ].…”

Function of Molybdenum Insertases

“…In the first step of this highly conserved pathway, GTP is converted to cyclic pyranopterin monophosphate [ 15 , 16 , 17 , 18 ], which in the following second step is converted to molybdopterin (MPT, [ 19 , 20 , 21 ]. In the third step of Moco biosynthesis, MPT is adenylylated by the molybdenum (Mo)-insertase G-domain, yielding MPT-AMP (termed adenylated MPT, [ 22 , 23 ]) and in the following fourth step, molybdate is inserted into MPT-AMP by the E-domain, yielding Moco-AMP (termed adenylated Moco [ 14 ]). Subsequently, the Moco-AMP phosphoanydrid bond is hydrolyzed and mature Moco is liberated from its synthesizing enzyme ([ 14 ], Figure 2 ).…”

“…In the third step of Moco biosynthesis, MPT is adenylylated by the molybdenum (Mo)-insertase G-domain, yielding MPT-AMP (termed adenylated MPT, [ 22 , 23 ]) and in the following fourth step, molybdate is inserted into MPT-AMP by the E-domain, yielding Moco-AMP (termed adenylated Moco [ 14 ]). Subsequently, the Moco-AMP phosphoanydrid bond is hydrolyzed and mature Moco is liberated from its synthesizing enzyme ([ 14 ], Figure 2 ).…”