Purification and properties of β-lysine mutase, a pyridoxal phosphate and B12 coenzyme dependent enzyme

Baker, John J.; Drift, Chris van der; Stadtman, Thressa C.

doi:10.1021/bi00730a006

Cited by 56 publications

(51 citation statements)

References 19 publications

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“…Baker et al (4) reported that AdoCbldependent ␤-lysine mutase undergoes concomitant inactivation during catalysis that accompanies irreversible cleavage of the Co-C bond of AdoCbl. They noted that this inactivation is prevented by the addition of ATP and a sulfhydryl protein named E2.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

et al. 2004

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The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg 2؉ , and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5 end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O 2 -inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg 2؉ . The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.Certain enzymes utilize the high reactivity of free radicals to catalyze the reactions that are difficult to catalyze by ionic mechanisms (6). Since the radicals involved in the catalysis are highly reactive, radical enzymes have a general tendency to undergo suicide inactivation during catalysis or inactivation by oxygen (40). Therefore, protection of the radical intermediates against undesired side reactions or reactivation of the inactivated enzymes is of essential importance for radical-catalyzed enzymatic reactions.Adenosylcobalamin (AdoCbl)-dependent enzymes are known to catalyze reactions including carbon skeleton rearrangements, heteroatom eliminations, and intramolecular amino group migrations by radical mechanisms (5, 13). An adenosyl radical formed by homolytic cleavage of the Co-C bond of enzyme-bound AdoCbl is directly involved in catalysis. The holoenzymes of diol dehydratase and glycerol dehydratase undergo suicide inactivation by glycerol during catalysis (3,29,30,44) or O 2 inactivation in the absence of substrate (25,30,38,47). This phenomenon is of special interest because glycerol is a physiological substrate for these enzymes (1,17,18,41,42). This apparent inconsistency was solved by previous findings that the glycerol-inactivated enzymes in permeabilized cells (in situ) of Klebsiella oxytoca and Klebsiella pneumoniae undergo rapid reactivation in the presence of ATP, Mg 2ϩ (or Mn 2ϩ ), and AdoCbl (20,46). ...

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Section: Discussionmentioning

confidence: 99%

Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

et al. 2004

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“…Both FN and FNV can degrade either L-lysine or D-lysine, like Clostridium spp and P. putida spp oleovorans. The operon encoding enzymes for lysine utilization is similar to that of P. gingivalis, including lysine permease, lysine 2,3-aminomutase, and a bifunctional D-lysine 5,6-aminomutase gene, like that of Clostridium sticklandii (Baker et al 1973;Chang and Frey 2000).…”

Section: Dna Modificationmentioning

confidence: 99%

Genome Analysis of F. nucleatum sub spp vincentii and Its Comparison With the Genome of F. nucleatum ATCC 25586

Kapatral¹,

Ivanova²,

Anderson³

et al. 2003

Genome Res.

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We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp. vincentii (FNV), and compare that genome with F. nucleatum ATCC 25586 (FN). A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified. Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms. In addition to the excretion of butyrate, H 2 S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate. Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen. It appears to transport ferrous iron by an anaerobic ferrous transporter. Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV. ORFs for ethanolamine utilization, thermostable carboxypeptidase, ␥ glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV. FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present. These capabilities allow Fusobacteria to survive in a mixed culture in the mouth.

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“…Section 1734 solely to indicate this fact. tation of L-ornithine (12) by interconverting D-ornithine to 2,4-diaminopentanoic acid (13). OAM is a ␣ 2 ␤ 2 heterodimer comprising two strongly associating subunits, OraS (12.8 kDa) and OraE (82.9 kDa) (13).…”

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confidence: 99%

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Wolthers

Rigby

Scrutton

2008

Journal of Biological Chemistry

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We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin ( ; coenzyme B 12 ) serves as a radical repository for a group of enzymes that catalyze unusual isomerizations, whereby a hydrogen atom (H) is interchanged with an electron-withdrawing group (X) on a neighboring carbon atom (Scheme 1) (1-4). To date, 11 AdoCbl-dependent isomerases have been identified and grouped into three classes: (i) Class I (mutases) that catalyze carbon skeletal rearrangements; (ii) Class II (eliminases) that catalyze, with one exception, the migration and elimination of a heteroatom; and (iii) Class III (aminomutases) that catalyze intramolecular 1,2-amino shifts. Turnover for all AdoCbl-dependent isomerases begins with substrate-induced homolysis of the AdoCbl Co-C bond and formation of two paramagnetic centers: the 5Ј-deoxyadenosyl radical (Ado ⅐ ) and cob(II)alamin. The highly reactive Ado ⅐ species propagates radical formation by abstracting a hydrogen atom from the substrate (or an amino acid side chain in the case of ribonucleotide reductase) (5), generating deoxyadenosine and a substrate radical. The latter carbon-centered radical isomerizes to a product radical intermediate, which then reabstracts a hydrogen atom to form Ado ⅐ . Geminate recombination between Ado ⅐ and cob(II)alamin regenerates AdoCbl and primes the enzyme for another catalytic cycle.Studies on AdoCbl-dependent isomerases have shown that the first step in the catalytic cycle (homolytic rupture of the Co-C bond) is coupled kinetically to hydrogen abstraction by Ado ⅐ (6 -9). Thus, the highly reactive Ado ⅐ species is short lived due to rapid neutralization by hydrogen abstraction, and this species has yet to be observed. The second paramagnetic center, cob(II)alamin, "lingers" in the active site, until product is formed, after which it recombines with Ado ⅐ to form the resting enzyme. In the presence of substrate, cob(II)alamin accumulates at a steady-state concentration, which can be observed by EPR and UV-visible spectroscopy. Class I and Class II isomerases have been extensively studied, and the cob(II)alamin spectroscopic signature has been valuable in studies of mechanism (6,8,10). Lysine 5,6-aminomutase (5,6-LAM) is the only Class III enzyme for which detailed studies of mechanism are reported. Cob(II)alamin does not accumulate in steady-state turnover as the enzyme undergoes rapid "suicide inactivation" involving removal of an electron from cob(II)alamin by a substrate and/or product radical intermediate (11). Irreversible formation of cob(III)alamin results, and cob(II)alamin is unable to recombine with Ado ⅐ to reform AdoCbl in the resting form of the enzyme.

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Purification and properties of β-lysine mutase, a pyridoxal phosphate and B12 coenzyme dependent enzyme

Cited by 56 publications

References 19 publications

Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

Genome Analysis of F. nucleatum sub spp vincentii and Its Comparison With the Genome of F. nucleatum ATCC 25586

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

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