Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependentd-Ornithine Aminomutase from Clostridium sticklandii

Chen, Hao‐Ping; Wu, Shih‐Hsiung; Lin, Ya-Lin; Chen, Chih‐Ming; Tsay, San-San

doi:10.1074/jbc.m108365200

Cited by 52 publications

(37 citation statements)

References 24 publications

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“…During steady-state turnover of AdoCbl-dependent enzymes, these generally correspond to the low spin Co 2ϩ atom of cob(II)alamin and a substrate/product-like radical intermediate. Magnetic interactions between Co 2ϩ and the organic radical create mutual perturbations in the EPR spectra of the component radicals, which provide information on the level of spin-spin coupling between the paramagnetic centers and hence their relative positions (13). The EPR spectrum of the holoenzyme mixed with D-ornithine did not show any evidence of a paramagnetic species; the EPR spectrum closely resembled the control sample, which contained only the holoenzyme and is essentially a base-line spectrum (data not shown).…”

Section: Epr Spectroscopic Characterization Of Holo-oam-eprmentioning

confidence: 99%

“…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). The enzyme contains AdoCbl and PLP (12), and both are required in the catalytic mechanism (Fig.…”

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confidence: 99%

“…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%

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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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Section: Epr Spectroscopic Characterization Of Holo-oam-eprmentioning

confidence: 99%

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confidence: 99%

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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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“…Although the proteins of this oxidative pathway were characterized biochemically 30 years ago for C. sticklandii, only the genes corresponding to the two subunits of OA (oraS and oraE) have been identified to date (3). In this article, we present the analysis of genes which are colocalized with oraS and oraE and which are hypothesized to be involved in the conversion of L-ornithine to D-ornithine, the oxidative deamination of DAP, and the thiolytic cleavage of AKP.…”

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confidence: 99%

“…D-Ornithine is next converted to (2R,4S)-2,4-diaminopentanoate (DAP) through the action of D-ornithine aminomutase (OA) (EC 5.4.3.5) (Fig. 1, step 2), an adenosylcobalamine and pyridoxal phosphate (PLP)-dependent enzyme (3,14). DAP then undergoes a NAD ϩ -or NADP ϩ -dependent oxidative deamination by DAP dehydrogenase (DAPDH) (EC 1.4.1.12) (Fig.…”

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A Conserved Gene Cluster Rules Anaerobic Oxidative Degradation of l -Ornithine

et al. 2009

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For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode L-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.

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Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

Pang

Scrutton

Sutcliffe

2014

Chemistry A European J

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We present here our computational study of the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Using both model systems and a combined QM/MM approach, our calculations suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen (N1) and the Schiff base nitrogen (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amine groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate in order to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions.3

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Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependentd-Ornithine Aminomutase from Clostridium sticklandii

Cited by 52 publications

References 24 publications

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

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

A Conserved Gene Cluster Rules Anaerobic Oxidative Degradation of l -Ornithine

Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

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