The Crystal Structure of Escherichia coli MoeA and Its Relationship to the Multifunctional Protein Gephyrin

Xiang, Song; Nichols, Jason M.; Rajagopalan, K.V.; Schindelin, Hermann

doi:10.1016/s0969-2126(01)00588-3

Cited by 101 publications

(129 citation statements)

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“…Biochemical studies have indicated that newly formed MPT remains tightly bound to the MoaD-MoaE complex (MPT synthase) until its transfer to proteins able to bind it with higher affinity (13). MogA and MoeA proteins constitute such candidates and have been shown to bind MPT with distinct affinities (6,11,14). MPT might then be transferred from MPT synthase to MogA by direct protein interaction, and an activated molybdenum species can be subsequently inserted into the MogAbound MPT by the aid of MoeA.…”

mentioning

confidence: 99%

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Magalon

Frixon

Pommier

et al. 2002

Journal of Biological Chemistry

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The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.

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mentioning

confidence: 99%

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Magalon

Frixon

Pommier

et al. 2002

Journal of Biological Chemistry

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“…The orientation of domain II was more variable (See Fig. 7, and Supplemental Table III), as was the case for the two wild type structures (19). Domain II variability was most pronounced in the T100A and R137Q variants, where domain II of one monomer of each was poorly defined in the electron density maps.…”

Section: Structural Studies Of Moea Variantsmentioning

confidence: 82%

“…Using the Transformer Site-Directed Mutagenesis Kit (BD/Clontech), site-directed mutagenesis of conserved MoeA residues was performed on pJNeA11, which contains the E. coli moeA gene in a pET11a expression vector (Novagen) (19). Substitutions were made at residues Asp59, Thr100, Arg137, Asp142, Glu188, Asp228, Asp259, Lys275, Lys279, Arg350, and Ser371 using 5′-phosphorylated mutagenic primers overlapping the codon to be modified.…”

Section: Mutagenesis Of Moeamentioning

confidence: 99%

“…All MoeA variants were purified using the method previously described for wild-type MoeA (19) except that the proteins were expressed in the AH69(DE3) strain and cells were lysed in the presence of 1 μg/ml leupeptin and 1 mM benzamidine-HCl. With the exception of T100W, S371W, and Δ-MoeA, protein concentrations were calculated using the ∈ 280 of 0.61 (mg/ ml) −1 cm −1 calculated for wild-type MoeA (19). The T100W and S371W variants were quantitated using the BCA protein assay (Pierce), while Δ-MoeA was quantitated using a calculated (based upon primary sequence) ∈ 280 of 0.71 (mg/ml) −1 cm −1 or 24,390 M −1 cm −1 .…”

Section: Mutagenesis Of Moeamentioning

confidence: 99%

“…With the exception of T100A, R137Q and D142N, all variants crystallized in space group P2 1 2 1 2 1 with cell dimensions roughly corresponding to either of the two crystal forms described for wild type MoeA (19), with two MoeA monomers per asymmetric unit (Supplemental Table I). The T100A and D142N variants also crystallized in space group P2 1 2 1 2 1 , but with cell dimensions that are only distantly related to the NatI crystal form, and also contain two monomers in the asymmetric unit.…”

Section: X-ray Crystallographymentioning

confidence: 99%

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Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft^,

et al. 2006

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SUMMARYThe molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, that of metal ligation to molybdopterin (the organic component of the cofactor) to form active cofactor. In Escherichia coli, the MoeA protein mediates Mo ligation to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The xray crystal structures for both proteins have been previously described as well as two essential MogA residues; Asp49 and Asp82. Here we describe the detailed mutational analysis of the MoeA protein.Variants of conserved residues at the putative active site of MoeA were analyzed for loss of function in two different, previously described assays; one employing moeA − crude extracts, and the other utilizing a defined system. Oddly, no correlation was observed between the observed activity in the two assays. In fact, our results showed a general trend towards an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified components assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. Based upon these results, two different functional areas were assigned to regions at or near the MoeA active site cleft. Keywords molybdopterin; MPT; Moco; molybdenum; MoeA; MogA; gephyrin; Cnx1; cinnamon PDB IDs for the variants: 2NQK (D59N); 2NQM (T100A); 2NQN (T100W); 2NQQ (R137Q); 2NQR (D142N); 2NQS (E188A); 2NQU (E188Q); 2NQV (D228A); 2NRO (K279Q); 2NRP (R350A); and 2NRS (S371W) All molybdenum-containing enzymes with the exception of nitrogenase, utilize a molybdenum cofactor (Moco) 1 consisting of a mononuclear Mo atom coordinated via a cis-dithiolene moiety to the organic molecule molybdopterin (MPT). Moco-containing enzymes function as oxidoreductases in a myriad of reactions in carbon, nitrogen, and sulfur cycles (1). In † This work was supported by National Institutes of Health Grant GM00091 (to KVR) and DK 54835 (to HS). * To whom correspondence should be addressed, Phone: (919) Fax: (919) The X-ray crystal structures of E. coli MoeA has previously been solved, and a putative active site assigned (18,19). To gain a more thorough understanding of the mechanism of MoeAmediated molybdenum ligation, a detailed site-directed mutagenesis study of conserved residues at the putative MoeA active site was undertaken. These variants were analyzed for Moco binding and for loss of function in both the moeA − crude extract assay and the fullydefined system. Results from these experiments were utilized to provide the first picture of the distribution of function across the MoeA 3-D structure. MATERIALS AND METHODS Mutagenesis of MoeAUsing the Transformer Site-Directed Mutagenesi...

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Assembly ofMoco inMo/WEnzymes

Leimkühler

2017

Encyclopedia of Inorganic and Bioinorganic Chemistry

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The biosynthesis of the molybdenum cofactor (Moco) is ubiquitous and highly conserved in all organisms from bacteria to humans. In Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin‐based 6‐alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into three steps in eukaryotes, and four steps in bacteria and archaea: (i) the starting point is the formation of the cPMP from 5′GTP, (ii) in the second step MPT is formed by the insertion of two sulfur molecules into cPMP, (iii) in the third step the molybdenum atom is inserted into MPT and Moco is formed, and (iv) additional modification of Moco occurs in bacteria and archaea in a fourth step by the attachment of nucleotides (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. Further, small differences exist in Moco formation among the different phyla. In higher eukaryotes Moco biosynthesis is located in different cellular compartments, many individual Moco biosynthesis proteins appear to have several cellular roles, and some proteins are shared between different biosynthetic pathways. Further, bacteria contain a large variety of more than 60 different molybdoenzymes being involved in specific, however, usually nonessential redox reactions. In contrast, in humans only four different molybdoenzymes have been identified, and a defect in Moco biosynthesis is lethal due to the loss of sulfite oxidase activity. This review will focus on the biosynthesis of Moco in bacteria and humans.

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The Crystal Structure of Escherichia coli MoeA and Its Relationship to the Multifunctional Protein Gephyrin

Cited by 101 publications

References 45 publications

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft^,

Assembly ofMoco inMo/WEnzymes

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The Crystal Structure of Escherichia coli MoeA and Its Relationship to the Multifunctional Protein Gephyrin

Cited by 101 publications

References 45 publications

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft,

Assembly ofMoco inMo/WEnzymes

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Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft^,