A Protein Pre-Organized to Trap the Nucleotide Moiety of Coenzyme B12: Refined Solution Structure of the B12-Binding Subunit of Glutamate Mutase from Clostridium tetanomorphum

Adenosylcobalamin-dependent glutamate mutase (EC 5.4.99.1) from Clostridium tetanomorphum comprises two protein components, MutE and MutS. The formation of the holoenzyme is a kinetically complex process that involves the co-operative association of MutS, MutE and adenosylcobalamin. The MutS portion of the cobalamin-binding site is conserved within a group of adenosylcobalamin-dependent enzymes that catalyse similar isomerizations. However, in contrast with glutamate mutase, in these other enzymes the cobalamin-binding region represented by MutS is present as a C-terminal domain. We have investigated the effect on the structural and kinetic properties of glutamate mutase of linking MutS to the C-terminus of MutE. Kinetic analysis of this protein, MutES, showed, unexpectedly, that enzyme activity was still co-operatively dependent on protein concentration. The Km for l-glutamate was unchanged from the wild type, whereas Vmax was decreased to approx. one-thirtieth and the Km for coenzyme increased approx. 10-fold. Investigation of the quaternary structure of MutES by equilibrium ultracentrifugation indicated that the protein existed in equilibrium between monomeric and dimeric forms. Thus linking MutE and MutS together seems to substantially weaken the contacts that are responsible for the dimerization of MutE. The two domains of the MutES monomer seem unable to communicate, so that active enzyme is formed by the intermolecular association of two MutES subunits in a co-operative manner.

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Conversion of Vitamin B12 to Coenzyme B12 in Cell-free Extracts of Clostridium tetanomorphum

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)64145-6 ◽

1961 ◽

Vol 236 (7) ◽

pp. PC40-PC42 ◽

Cited By ~ 4

Author(s):

Herbert Weissbach ◽

Betty Redfield ◽

Alan Peterkofsky

Keyword(s):

Vitamin B12 ◽

Coenzyme B12 ◽

Clostridium Tetanomorphum

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Molecular Modeling Studies of the Conserved B12-Binding Motif and Its Variants from Clostridium tetanomorphum Glutamate Mutase

Protein and Peptide Letters ◽

10.2174/092986610791190264 ◽

2010 ◽

Vol 17 (6) ◽

pp. 759-764 ◽

Cited By ~ 1

Author(s):

Chun-Hua Hsu ◽

Hao-Ping Chen

Keyword(s):

Molecular Modeling ◽

Binding Motif ◽

Glutamate Mutase ◽

Modeling Studies ◽

Clostridium Tetanomorphum ◽

Molecular Modeling Studies

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Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA

Nucleic Acids Research ◽

10.1093/nar/gkaa751 ◽

2020 ◽

Vol 48 (19) ◽

pp. 11146-11161

Author(s):

Naining Xu ◽

Yingying You ◽

Changdong Liu ◽

Maxim Balasov ◽

Lee Tung Lun ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Solution Structure ◽

Origin Recognition Complex ◽

Structural Basis ◽

Dna Replication Origin ◽

Dna Alterations ◽

Replication Initiator ◽

Binding Subunit ◽

Origin Recognition

Abstract The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

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Radical Shuttling in a Protein: Ribose Pseudorotation Controls Alkyl-Radical Transfer in the Coenzyme B12 Dependent Enzyme Glutamate Mutase

Angewandte Chemie International Edition ◽

10.1002/1521-3773(20010917)40:18<3377::aid-anie3377>3.0.co;2-8 ◽

2001 ◽

Vol 40 (18) ◽

pp. 3377-3380 ◽

Cited By ~ 77

Author(s):

Karl Gruber ◽

Riikka Reitzer ◽

Christoph Kratky

Keyword(s):

Alkyl Radical ◽

Coenzyme B12 ◽

Glutamate Mutase ◽

Radical Transfer

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Coenzyme B12 dependent glutamate mutase

Current Opinion in Chemical Biology ◽

10.1016/s1367-5931(02)00368-x ◽

2002 ◽

Vol 6 (5) ◽

pp. 598-603 ◽

Cited By ~ 29

Author(s):

Karl Gruber ◽

Christoph Kratky

Keyword(s):

Coenzyme B12 ◽

Glutamate Mutase

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Protein–coenzyme interactions in adenosylcobalamin-dependent glutamate mutase

Biochemical Journal ◽

10.1042/bj3550131 ◽

2001 ◽

Vol 355 (1) ◽

pp. 131-137 ◽

Cited By ~ 1

Author(s):

Marja S. HUHTA ◽

Hao-Ping CHEN ◽

Craig HEMANN ◽

C. Russ HILLE ◽

E. Neil G. MARSH

Keyword(s):

Gel Filtration ◽

Visible Spectrum ◽

Titration Calorimetry ◽

Coenzyme B12 ◽

Glutamate Mutase ◽

Carbon Bond ◽

Coenzyme Binding ◽

Radical Intermediates ◽

Bond Stretching ◽

Uv Visible

Glutamate mutase catalyses an unusual isomerization involving free-radical intermediates that are generated by homolysis of the cobalt–carbon bond of the coenzyme adenosylcobalamin (coenzyme B12). A variety of techniques have been used to examine the interaction between the protein and adenosylcobalamin, and between the protein and the products of coenzyme homolysis, cob(II)alamin and 5′-deoxyadenosine. These include equilibrium gel filtration, isothermal titration calorimetry, and resonance Raman, UV-visible and EPR spectroscopies. The thermodynamics of adenosylcobalamin binding to the protein have been examined and appear to be entirely entropy-driven, with ∆S = 109 Jċmol-1ċK-1. The cobalt–carbon bond stretching frequency is unchanged upon coenzyme binding to the protein, arguing against a ground-state destabilization of the cobalt–carbon bond of adenosylcobalamin by the protein. However, reconstitution of the enzyme with cob(II)alamin and 5′-deoxyadenosine, the two stable intermediates formed subsequent to homolysis, results in the blue-shifting of two of the bands comprising the UV-visible spectrum of the corrin ring. The most plausible interpretation of this result is that an interaction between the protein, 5′-deoxyadenosine and cob(II)alamin introduces a distortion into the ring corrin that perturbs its electronic properties.

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