NORMAL GENOMIC

• Bader G, Gomez-Ortiz M, Haussmann C, Bacher A, Huber R, Fischer M
• Structure of the molybdenum-cofactor biosynthesis protein MoaB ofEscherichia coli.
• Acta Crystallogr D Biol Crystallogr. 2004; 60: 1068-75
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• The moaABC operon of Escherichia coli is involved in early steps of thebiosynthesis of the molybdenum-binding cofactor molybdopterin, but theprecise functions of the cognate proteins are not known. The crystalstructure of the MoaB protein from E. coli was determined by multipleanomalous dispersion at 2.1 angstroms A resolution and refined to an Rfactor of 20.4% (Rfree = 25.0%). The protein is a 32-symmetric hexamer,with the monomers consisting of a central beta-sheet flanked by helices onboth sides. The overall fold of the monomer is similar to those of theMogA protein of E. coli, the G-domains of rat and human gephyrin and theG-domains of Cnx1 protein from A. thaliana, all of which are involved inthe insertion of an unknown molybdenum species into molybdopterin to formthe molybdenum cofactor. Furthermore, the MoaB protein shows significantsequence similarity to the cinnamon protein from Drosophila melanogaster.In addition to other functions, all these proteins are involved in thebiosynthesis of the molybdenum cofactor and have been shown to bindmolybdopterin. The close structural homology to MogA and the gephyrin andCnx1 domains suggests that MoaB may bind a hitherto unidentified pterincompound, possibly an intermediate in molybdopterin biosynthesis.

• Sanishvili R et al.
• The crystal structure of Escherichia coli MoaB suggests a probable role inmolybdenum cofactor synthesis.
• J Biol Chem. 2004; 279: 42139-46
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• The crystal structure of Escherichia coli MoaB was determined bymultiwavelength anomalous diffraction phasing and refined at 1.6-Aresolution. The molecule displayed a modified Rossman fold. MoaB isassembled into a hexamer composed of two trimers. The monomers have highstructural similarity with two proteins, MogA and MoeA, from themolybdenum cofactor synthesis pathway in E. coli, as well as with domainsof mammalian gephyrin and plant Cnx1, which are also involved inmolybdopterin synthesis. Structural comparison between these proteins andthe amino acid conservation patterns revealed a putative active site inMoaB. The structural analysis of this site allowed to advance severalhypothesis that can be tested in further studies.

• Schwarz G, Schrader N, Mendel RR, Hecht HJ, Schindelin H
• Crystal structures of human gephyrin and plant Cnx1 G domains: comparativeanalysis and functional implications.
• J Mol Biol. 2001; 312: 405-18
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• The molybdenum cofactor (Moco) consists of a unique and conserved pterinderivative, usually referred to as molybdopterin (MPT), which coordinatesthe essential transition metal molybdenum (Mo). Moco is required for theenzymatic activities of all Mo-enzymes, with the exception of nitrogenaseand is synthesized by an evolutionary old multi-step pathway that isdependent on the activities of at least six gene products. In eukaryotes,the final step of Moco biosynthesis, i.e. transfer and insertion of Mointo MPT, is catalyzed by the two-domain proteins Cnx1 in plants andgephyrin in mammals. Gephyrin is ubiquitously expressed, and was initiallyfound in the central nervous system, where it is essential for clusteringof inhibitory neuroreceptors in the postsynaptic membrane. Gephyrin andCnx1 contain at least two functional domains (E and G) that are homologousto the Escherichia coli proteins MoeA and MogA, the atomic structures ofwhich have been solved recently. Here, we present the crystal structuresof the N-terminal human gephyrin G domain (Geph-G) and the C-terminalArabidopsis thaliana Cnx1 G domain (Cnx1-G) at 1.7 and 2.6 A resolution,respectively. These structures are highly similar and compared to MogAreveal four major differences in their three-dimensional structures: (1)In Geph-G and Cnx1-G an additional alpha-helix is present between thefirst beta-strand and alpha-helix of MogA. (2) The loop between alpha 2and beta 2 undergoes conformational changes in all three structures. (3) Abeta-hairpin loop found in MogA is absent from Geph-G and Cnx1-G. (4) TheC terminus of Geph-G follows a different path from that in MogA. Based onthe structures of the eukaryotic proteins and their comparisons with E.coli MogA, the predicted binding site for MPT has been further refined. Inaddition, the characterized alternative splice variants of gephyrin areanalyzed in the context of the three-dimensional structure of Geph-G.

• Xiang S, Nichols J, Rajagopalan KV, Schindelin H
• The crystal structure of Escherichia coli MoeA and its relationship to themultifunctional protein gephyrin.
• Structure. 2001; 9: 299-310
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• BACKGROUND: Molybdenum cofactor (Moco) biosynthesis is an evolutionarilyconserved pathway present in archaea, eubacteria, and eukaryotes. Inhumans, genetic abnormalities in the biosynthetic pathway result in Mocodeficiency, which is accompanied by severe neurological symptoms and deathshortly after birth. The Escherichia coli MoeA and MogA proteins areinvolved in the final step of Moco biosynthesis: the incorporation ofmolybdenum into molybdopterin (MPT), the organic pyranopterin moiety ofMoco. RESULTS: The crystal structure of E. coli MoeA has been refined at 2A resolution and reveals that the highly elongated MoeA monomer consistsof four clearly separated domains, one of which is structurally related toMogA, indicating a divergent evolutionary relationship between bothproteins. The active form of MoeA is a dimer, and a putative active siteappears to be localized to a cleft formed between domain II of the firstmonomer and domains III and IV of the second monomer. CONCLUSIONS: Ineukaryotes, MogA and MoeA are fused into a single polypeptide chain. Thecorresponding mammalian protein gephyrin has also been implicated in theanchoring of glycinergic receptors to the cytoskeleton at inhibitorysynapses. Based on the structures of MoeA and MogA, gephyrin is surmisedto be a highly organized molecule containing at least five domains. Thismultidomain arrangement could provide a structural basis for itsfunctional diversity. The oligomeric states of MoeA and MogA suggest howgephyrin could assemble into a hexagonal scaffold at inhibitory synapses.

• Liu MT, Wuebbens MM, Rajagopalan KV, Schindelin H
• Crystal structure of the gephyrin-related molybdenum cofactor biosynthesisprotein MogA from Escherichia coli.
• J Biol Chem. 2000; 275: 1814-22
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• Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conservedpathway in archaea, eubacteria, and eukaryotes, including humans. Geneticdeficiencies of enzymes involved in this biosynthetic pathway trigger anautosomal recessive disease with severe neurological symptoms, whichusually leads to death in early childhood. The MogA protein exhibitsaffinity for molybdopterin, the organic component of Moco, and has beenproposed to act as a molybdochelatase incorporating molybdenum into Moco.MogA is related to the protein gephyrin, which, in addition to its role inMoco biosynthesis, is also responsible for anchoring glycinergic receptorsto the cytoskeleton at inhibitory synapses. The high resolution crystalstructure of the Escherichia coli MogA protein has been determined, and itreveals a trimeric arrangement in which each monomer contains a central,mostly parallel beta-sheet surrounded by alpha-helices on either side.Based on structural and biochemical data, a putative active site wasidentified, including two residues that are essential for the catalyticmechanism.

• Ramming M, Kins S, Werner N, Hermann A, Betz H, Kirsch J
• Diversity and phylogeny of gephyrin: tissue-specific splice variants, genestructure, and sequence similarities to molybdenum cofactor-synthesizingand cytoskeleton-associated proteins.
• Proc Natl Acad Sci U S A. 2000; 97: 10266-71
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• Gephyrin is essential for both the postsynaptic localization of inhibitoryneurotransmitter receptors in the central nervous system and thebiosynthesis of the molybdenum cofactor (Moco) in different peripheralorgans. Several alternatively spliced gephyrin transcripts have beenidentified in rat brain that differ in their 5' coding regions. Here, wedescribe gephyrin splice variants that are differentially expressed innon-neuronal tissues and different regions of the adult mouse brain.Analysis of the murine gephyrin gene indicates a highly mosaicorganization, with eight of its 29 exons corresponding to thealternatively spliced regions identified by cDNA sequencing. The N- andC-terminal domains of gephyrin encoded by exons 3-7 and 16-29,respectively, display sequence similarities to bacterial, invertebrate,and plant proteins involved in Moco biosynthesis, whereas the centralexons 8, 13, and 14 encode motifs that may mediate oligomerization andtubulin binding. Our data are consistent with gephyrin having evolved froma Moco biosynthetic protein by insertion of protein interaction sequences.

• Stallmeyer B et al.
• The neurotransmitter receptor-anchoring protein gephyrin reconstitutesmolybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells.
• Proc Natl Acad Sci U S A. 1999; 96: 1333-8
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• The molybdenum cofactor (Moco), a highly conserved pterin compoundcomplexing molybdenum, is required for the enzymatic activities of allmolybdenum enzymes except nitrogenase. Moco is synthesized by a unique andevolutionarily old pathway that requires the activities of at least sixgene products. Some of the proteins involved in bacterial, plant, andinvertebrate Moco biosynthesis show striking homologies to the primarystructure of gephyrin, a polypeptide required for the clustering ofinhibitory glycine receptors in postsynaptic membranes in the rat centralnervous system. Here, we show that gephyrin binds with high affinity tomolybdopterin, the metabolic precursor of Moco. Furthermore, gephyrinexpression can reconstitute Moco biosynthesis in Moco-deficient bacteria,a molybdenum-dependent mouse cell line, and a Moco-deficient plant mutant.Conversely, inhibition of gephyrin expression by antisense RNA expressionin cultured murine cells reduces their Moco content significantly. Thesedata indicate that in addition to clustering glycine receptors, gephyrinalso is involved in Moco biosynthesis and illustrate the remarkableconservation of its function in Moco biosynthesis throughout phylogeny.

• Stallmeyer B, Nerlich A, Schiemann J, Brinkmann H, Mendel RR
• Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1encodes a multifunctional two-domain protein homologous to a mammalianneuroprotein, the insect protein Cinnamon and three Escherichia coliproteins.
• Plant J. 1995; 8: 751-62
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• The molybdenum co-factor (Moco) is an essential part of all eukaryoticmolybdoenzymes. It is a molybdopterin and reveals the same principalstructure in eubacteria, archaebacteria and eukaryotes. This paper reportsthe isolation of cnx1, a cDNA clone of Arabidopsis thaliana whichcomplements the Escherichia coli Moco mutant mogA. The mapping data ofthis cDNA correlate well with the mapping position of the A. thalianamolybdenum co-factor locus chl6. As mutants in chl6 are known to berepairable by high concentrations of molybdate, the defective gene is verylikely to be involved in the last step of Moco biosynthesis, that is, theinsertion of molybdenum into molybdopterin. The protein encoded by cnx1shows a two-domain structure: the N-terminal domain is homologous to theE. coli Moco protein MoeA, the C-terminal domain is homologous to the E.coli Moco proteins MoaB and MogA, respectively. These homologies show thatpart of the prokaryotic Moco biosynthetic pathway accomplished bymonofunctional proteins in E. coli, is performed by a singlemultifunctional protein in eukaryotes. In addition Cnx1 is homologous tothe eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, aDrosophila protein with a function in Moco biosynthesis. These proteinsalso show a two-domain structure but the order of the domains is inversedas compared with Cnx1. Southern analysis indicates the existence of atleast one further member, in addition to the cnx1 gene, of this novel genefamily in the Arabidopsis genome.

• Kamdar KP, Shelton ME, Finnerty V
• The Drosophila molybdenum cofactor gene cinnamon is homologous to threeEscherichia coli cofactor proteins and to the rat protein gephyrin.
• Genetics. 1994; 137: 791-801
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• Essentially all organisms depend upon molybdenum oxidoreductases whichrequire a molybdopterin cofactor for catalytic activity. Mutationsresulting in a lack of the cofactor show a pleiotropic loss ofmolybdoenzyme activities and thereby define genes involved in cofactorbiosynthesis or utilization. In prokaryotes, two operons are directlyassociated with biosynthesis of the pterin moiety and its side chain whileadditional loci play a role in the acquisition of molybdenum and/oractivation of the cofactor. Here we report the cloning of cinnamon, aDrosophila molybdenum cofactor gene encoding a protein with sequencesimilarity to three of the prokaryotic cofactor proteins. In addition, theDrosophila cinnamon protein is homologous to gephyrin, a protein isolatedfrom the rat central nervous system. Our results suggest that someportions of the prokaryotic cofactor biosynthetic pathway composed ofmonofunctional proteins have evolved into a multifunctional protein inhigher eukaryotes.