A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

Toraya, Tetsuo; Mori, Kōichi

doi:10.1074/jbc.274.6.3372

Cited by 75 publications

(100 citation statements)

References 27 publications

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“…The molar absorption coefficient at 280 nm, calculated by the method of Gill and von Hippel (35) from the deduced amino acid composition and subunit structure for this enzyme, is 120,500 M Ϫ1 cm Ϫ1 (36).…”

Section: Methodsmentioning

confidence: 99%

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

Kawata

Kinoshita

Takahashi

et al. 2006

Journal of Biological Chemistry

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The importance of each active-site residue in adenosylcobalamindependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K ؉ was mutated to Ala or another amino acid residue. The E␣170A and D␣335A mutants were totally inactive, and the H␣143A mutant showed only a trace of activity, indicating that Glu-␣170, Asp-␣335, and His-␣143 are catalytic residues. The Q␣141A, Q␣296A, and S␣362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-␣296 is important for substrate binding and Gln-␣296 and Gln-␣141 for preventing the enzyme from mechanism-based inactivation. The E␣221A, E␣170H, and D␣335A did not form the (␣␤␥) 2 complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-␣170 and Asp-␣335 mutants, E␣170D and E␣170Q were 2.2 ؎ 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas D␣335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue ␣170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-␣170 and Asp-␣335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-␣170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.Diol dehydratase (DL-1,2-propanediol hydro-lyase, EC 4.2.1.28) is a coenzyme B 12 (AdoCbl) 2 -dependent enzyme that catalyzes the conversion of 1,2-propanediol, glycerol, and 1,2-ethanediol to the corresponding aldehydes (1-3). Labeling experiments of Rétey et al. (4,5) and Abeles and co-workers (6) as well as kinetic studies of this enzyme led to the general conclusion that, in the AdoCbl-dependent rearrangements, a hydrogen atom migrates from one carbon atom of substrate to an adjacent carbon atom in exchange for a group X that moves in the opposite direction (Reaction 1) (for reviews, see Refs. 7 and 8). In the case of diol dehydratase, REACTION 1 X is a hydroxyl group on C2 of 1,2-propanediol. A minimal mechanism was established in which the enzyme-bound 5Ј-deoxyadenosine serves as an intermediate in the hydrogen transfer process (Fig. 1A) (1, 7-11). It was proposed by theoretical calculation that the hydroxyl group on C2 migrates to C1 by a concerted pathway through a three-membered cyclic transition state (12). Other pathways can also be considered for the 1,2-shift of the hydroxyl group from the results of model reactions (13-16). The reaction pathway that we proposed for diol dehydratase is shown in Fig. 1B (1).The crystal structures of diol dehydratase in complexes with coenzyme analogs in the substrate-bound (17-19) and substrate-free (20) forms have been determined. The enzyme exists as a dimer of a heterotrimer (␣␤␥) 2 . The (␣␥) 2 complex a...

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Section: Methodsmentioning

confidence: 99%

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

Kawata

Kinoshita

Takahashi

et al. 2006

Journal of Biological Chemistry

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“…During DDH catalysis, the adenosyl group of Ado-B 12 is periodically lost due to catalytic by-reactions and replaced with an alternative upper ligand, usually a hydroxyl group, resulting in the formation of an inactive [OH-B 12 -DDH] complex (120,122). Reactivation begins with the PduGH reactivase, which converts the inactive [OH-B 12 -DDH] to free OH-B 12 and apoenzyme (120,(122)(123)(124)(125)(126)(127). Subsequently, Ado-B 12 spontaneously reassociates with apo-DDH to form an active holoenzyme, and the OH-B 12 is recycled back to Ado-B 12 by cobalamin reductase (PduS) and adenosyltransferase (PduO) (80,104,105) (Fig.…”

Section: Enzymes For Reactivation Of Diol Dehydratase and B 12 Recyclingmentioning

confidence: 99%

Diverse Bacterial Microcompartment Organelles

Chowdhury

Sinha

Chun

et al. 2014

Microbiol Mol Biol Rev

208

278

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SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.

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“…A corresponding reductase for MCM has not been found. B 12 -dependent eliminases like diol dehydratase use an ATP-dependent mechanism to exchange the oxidized inactive cofactor for an active one (15); however, such chaperones do not appear to be present in operons that encode MCM. Thus MCM does not appear to utilize the first two methods, leaving the third possibility that MeaB could play a role in cofactor protection during catalysis.…”

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confidence: 99%

Crystal Structure and Mutagenesis of the Metallochaperone MeaB

Hubbard¹,

Padovani

Labunska

et al. 2007

Journal of Biological Chemistry

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MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B 12 -dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central ␣/␤-core G domain that is typical of the GTPase family, as well as ␣-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP-and GTP-bound forms of this protein.Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.In the past few decades, an increasing number of guanine nucleotide-binding protein (G proteins) that act as chaperones in the assembly of target metalloenzymes have been described (1). These include UreG (2, 3), HypB (4, 5), CooC (6), and MeaB (7), which are involved in the metallocenter assembly of urease, NiFe-hydrogenase, CO dehydrogenase, and B 12 -dependent methylmalonyl-CoA mutase, respectively. Typically, G proteins act as molecular switches, with regions known as switch I and switch II undergoing large conformational changes upon GTP hydrolysis to communicate a signal. Although members of this metallochaperone G protein subfamily (called the G3E family) share appropriate sequence motifs (8) and exhibit low GTPase activity (3,4,6,9), their exact function with respect to target metalloenzymes remains to be determined. MeaB itself differs from the other G3E G proteins in that it possesses N-and C-terminal extensions of unknown function.MeaB is an auxiliary protein associated with methylmalonylCoA mutase (MCM) 2 (7, 9, 10), a coenzyme B 12 (adenosylcobalamin)-dependent enzyme that catalyzes the chemically challenging 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA using radical-based chemistry (11,12). A human orthologue of MeaB, MMAA, has been found to be the locus of mutations associated with type A (cblA) methylmalonic aciduria (MMA) (13), a rare congenital disease that manifests itself d...

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A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

Cited by 75 publications

References 27 publications

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

Diverse Bacterial Microcompartment Organelles

Crystal Structure and Mutagenesis of the Metallochaperone MeaB

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