Publications

• Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii.

  Chen H.P., Wu S.H., Lin Y.L., Chen C.M., Tsay S.S.

  D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. The oraS gene, which encodes a protein of ... >> More

  D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. The oraS gene, which encodes a protein of 121 amino acid residues with M(r) 12,800, is situated upstream of the oraE gene, which encodes a protein of 753 amino acid residues with M(r) 82,900. The holoenzyme appears to comprise a alpha(2)beta(2)-heterotetramer. OraS shows no significant homology to other proteins in the Swiss-Prot data base. The deduced amino acid sequence of OraE includes a conserved base-off/histidine-on cobalamin-binding motif, DXHXXG. OraE was expressed in E. coli as inclusion bodies. Refolding experiments on OraE indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate or adenosylcobalamin play important roles in refolding process. The K(m) values for d-ornithine, 5'-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5'-phosphate (PLP) are 44.5 +/- 2.8, 0.43 +/- 0.04, and 1.5 +/-0.1 microm, respectively; the k(cat) is 6.3 +/-0.1 s(-1). The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, and D-ornithine being present in the assay; no other cofactors were required. A red-shift in UV-visible absorption spectrum is observed when free adenosylcobinamide is bound by recombinant D-ornithine aminomutase and no significant change in spectrum when free adenosylcobinamide is bound by mutant OraE-H618G, demonstrating that the enzyme binds adenosylcobalamin in base-off/histidine-on mode. << Less

  J. Biol. Chem. 276:44744-44750(2001) [PubMed] [EuropePMC]

• Role of histidine 225 in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

  Makins C., Miros F.N., Scrutton N.S., Wolthers K.R.

  Pyridoxal 5'-phosphate (PLP), in the active site of ornithine 4,5-aminomutase (OAM), forms a Schiff base with N(δ) of the d-ornithine side chain and facilitates interconversion of the amino acid to (2R, 4S) 2,4-diaminopentanoic acid via a radical-based mechanism. The crystal structure of OAM revea ... >> More

  Pyridoxal 5'-phosphate (PLP), in the active site of ornithine 4,5-aminomutase (OAM), forms a Schiff base with N(δ) of the d-ornithine side chain and facilitates interconversion of the amino acid to (2R, 4S) 2,4-diaminopentanoic acid via a radical-based mechanism. The crystal structure of OAM reveals that His225 is within hydrogen bond distance to the PLP phenolic oxygen, and may influence the pK(a) of the Schiff base during radical rearrangement. To evaluate the role of His225 in radical stabilization and catalysis, the residue was substituted with a glutamine and alanine. The H225Q and H225A variants have a 3- and 10-fold reduction in catalytic turnover, respectively, and a decrease in catalytic efficiency (7-fold for both mutants). Diminished catalytic performance is not linked to an increase in radical-based side reactions leading to enzyme inactivation. pH-dependence studies show that k(cat) increases with the ionization of a functional group, but it is not attributed to His225. Binding of 2,4-diaminobutyric acid to native OAM leads to formation of an overstabilized 2,4-diaminobutyryl-PLP derived radical. In the H225A and the H225Q mutants, the radical forms and then decays, as evidenced by accumulation of cob(III)alamin. From these data, we propose that His225 enhances radical stability by acting as a hydrogen bond acceptor to the phenolic oxygen, which favors the deprotonated state of the imino nitrogen and leads to greater resonance stabilization of the 2,4-diaminobutyryl-PLP radical intermediate. The potential role of His225 in lowering the activation energy barrier to mediate PLP-dependent radical rearrangement is discussed. << Less

  Bioorg Chem 40:39-47(2012) [PubMed] [EuropePMC]

• Large-scale domain motions and pyridoxal-5'-phosphate assisted radical catalysis in coenzyme B12-dependent aminomutases.

  Maity A.N., Chen Y.H., Ke S.C.

  Lysine 5,6-aminomutase (5,6-LAM) and ornithine 4,5-aminomutase (4,5-OAM) are two of the rare enzymes that use assistance of two vitamins as cofactors. These enzymes employ radical generating capability of coenzyme B12 (5'-deoxyadenosylcobalamin, dAdoCbl) and ability of pyridoxal-5'-phosphate (PLP, ... >> More

  Lysine 5,6-aminomutase (5,6-LAM) and ornithine 4,5-aminomutase (4,5-OAM) are two of the rare enzymes that use assistance of two vitamins as cofactors. These enzymes employ radical generating capability of coenzyme B12 (5'-deoxyadenosylcobalamin, dAdoCbl) and ability of pyridoxal-5'-phosphate (PLP, vitamin B6) to stabilize high-energy intermediates for performing challenging 1,2-amino rearrangements between adjacent carbons. A large-scale domain movement is required for interconversion between the catalytically inactive open form and the catalytically active closed form. In spite of all the similarities, these enzymes differ in substrate specificities. 4,5-OAM is highly specific for D-ornithine as a substrate while 5,6-LAM can accept D-lysine and L-β-lysine. This review focuses on recent computational, spectroscopic and structural studies of these enzymes and their implications on the related enzymes. Additionally, we also discuss the potential biosynthetic application of 5,6-LAM. << Less

  Int J Mol Sci 15:3064-3087(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Large-scale domain conformational change is coupled to the activation of the Co-C bond in the B12-dependent enzyme ornithine 4,5-aminomutase: a computational study.

  Pang J., Li X., Morokuma K., Scrutton N.S., Sutcliffe M.J.

  We present here an energetic and atomistic description of how D-ornithine 4,5-aminomutase (OAM), an adenosylcobalamin (AdoCbl; coenzyme B(12))-dependent isomerase, employs a large-scale protein domain conformational change to orchestrate the homolytic rupture of the Co-C bond. Our results suggest ... >> More

  We present here an energetic and atomistic description of how D-ornithine 4,5-aminomutase (OAM), an adenosylcobalamin (AdoCbl; coenzyme B(12))-dependent isomerase, employs a large-scale protein domain conformational change to orchestrate the homolytic rupture of the Co-C bond. Our results suggest that in going from the open form (catalytically inactive) to the closed form (catalytically active), the Rossmann domain of OAM effectively approaches the active site as a rigid body. It undergoes a combination of a ~52° rotation and a ~14 Å translation to bring AdoCbl-initially positioned ~25 Å away-into the active-site cavity. This process is coupled to repositioning of the Ado moiety of AdoCbl from the eastern conformation to the northern conformation. Combined quantum mechanics and molecular mechanics calculations further indicate that in the open form, the protein environment does not impact significantly on the Co-C bond homolytic rupture, rendering it unusually stable, and thus catalytically inactive. Upon formation of the closed form, the Co-C bond is activated through the synergy of steric and electrostatic effects arising from tighter interactions with the surrounding enzyme. The more pronounced effect of the protein in the closed form gives rise to an elongated Co-C bond (by 0.03 Å), puckering of the ribose and increased "strain" energy on the Ado group and to a lesser extent the corrin ring. Our computational studies reveal novel strategies employed by AdoCbl-dependent enzymes in the control of radical catalysis. << Less

  J Am Chem Soc 134:2367-2377(2012) [PubMed] [EuropePMC]

• Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase.

  Makins C., Pickering A.V., Mariani C., Wolthers K.R.

  Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in ... >> More

  Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in OAM from Clostridium sticklandii and Glu392 in human MCM) to AdoCbl Co-C bond labilization and catalysis was evaluated by substituting the residue with a glutamine, aspartate, or alanine. The OAM variants, E338Q, E338D, and E338A, showed 90-, 380-, and 670-fold reductions in catalytic turnover and 20-, 60-, and 220-fold reductions in k(cat)/K(m), respectively. Likewise, the MCM variants, E392Q, E392D, and E392A, showed 16-, 330-, and 12-fold reductions in k(cat), respectively. Binding of substrate to OAM is unaffected by the single-amino acid mutation as stopped-flow absorbance spectroscopy showed that the rates of external aldimine formation in the OAM variants were similar to that of the native enzyme. The decrease in the level of catalysis is instead linked to impaired Co-C bond rupture, as UV-visible spectroscopy did not show detectable AdoCbl homolysis upon binding of the physiological substrate, d-ornithine. AdoCbl homolysis was also not detected in the MCM mutants, as it was for the native enzyme. We conclude from these results that a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for Co-C bond homolysis, thereby pointing to a key role for the conserved polar glutamate residue in controlling the initial generation of radical species. << Less

  Biochemistry 52:878-888(2013) [PubMed] [EuropePMC]

• Purification and properties of a pyridoxal phosphate and coenzyme B 12 dependent D-ornithine 5,4-aminomutase.

  Somack R., Costilow R.N.

  Biochemistry 12:2597-2604(1973) [PubMed] [EuropePMC]