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Crystal structure of putative CbiT from Methanocaldococcus jannaschii: an intermediate enzyme activity in cobalamin (vitamin B12) biosynthesis.

Padmanabhan B, Yokoyama S, Bessho Y - BMC Struct. Biol. (2013)

Bottom Line: The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum.The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity.The putative function of MJ0391 is discussed, based on structural homology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biophysics, National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore 560029, India. paddy@nimhans.kar.nic.in

ABSTRACT

Background: In the anaerobic pathway of cobalamin (vitamin B12) synthesis, the CbiT enzyme plays two roles, as a cobalt-precorrin-7 C15-methyltransferase and a C12-decarboxylase, to produce the intermediate, cobalt-precorrin 8.

Results: The primary structure of the hypothetical protein MJ0391, from Methanocaldococcus jannaschii, suggested that MJ0391 is a putative CbiT. Here, we report the crystal structure of MJ0391, solved by the MAD procedure and refined to final R-factor and R-free values of 19.8 & 27.3%, respectively, at 2.3 Å resolution. The asymmetric unit contains two NCS molecules, and the intact tetramer generated by crystallographic symmetry may be functionally important. The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum.

Conclusions: The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity. The putative function of MJ0391 is discussed, based on structural homology.

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Comparison of MJ0391 with MT0146/CbiT. (a) Stereo view of the superposition of MJ0391 (light green) on MT0146/CbiT (red), for all Cα-atoms corresponding to one subunit of the MT0146/CbiT tetramer (PDB ID: 1L3I). (b) The AdoHcy binding site region (stereo view). The interacting residues and the AdoHcy molecule (shown as ‘SAH’ in the figure) of the MT0146/CbiT complex are colored grey & cyan, respectively. The equivalent predicted interacting residues in MJ0391 are yellow. The Tyr65 residue is modeled as Ala, since the electron density corresponding to its side chain was not visible. (c) Electrostatic surface potential of the MJ0391 tetramer. The surfaces are colored blue and red for positive and negative electrostatic surface potentials, denoting less than -15 and greater than +15 KbT, respectively, where Kb is the Boltzmann constant and T is the temperature. The yellow and green arrows indicate the putative precorrin and AdoMet binding pockets, respectively. The surface potential was calculated with the software APBS[27] incorporated in the PyMOL program (Schrödinger, LLC.).
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Figure 4: Comparison of MJ0391 with MT0146/CbiT. (a) Stereo view of the superposition of MJ0391 (light green) on MT0146/CbiT (red), for all Cα-atoms corresponding to one subunit of the MT0146/CbiT tetramer (PDB ID: 1L3I). (b) The AdoHcy binding site region (stereo view). The interacting residues and the AdoHcy molecule (shown as ‘SAH’ in the figure) of the MT0146/CbiT complex are colored grey & cyan, respectively. The equivalent predicted interacting residues in MJ0391 are yellow. The Tyr65 residue is modeled as Ala, since the electron density corresponding to its side chain was not visible. (c) Electrostatic surface potential of the MJ0391 tetramer. The surfaces are colored blue and red for positive and negative electrostatic surface potentials, denoting less than -15 and greater than +15 KbT, respectively, where Kb is the Boltzmann constant and T is the temperature. The yellow and green arrows indicate the putative precorrin and AdoMet binding pockets, respectively. The surface potential was calculated with the software APBS[27] incorporated in the PyMOL program (Schrödinger, LLC.).

Mentions: Although the overall tertiary structures of the MJ0391 and MT0146/CbiT proteins are essentially similar, striking differences exist in the TH region, an important element for forming the tetrameric arrangements (Figure 4a). The dihedral angles at the equivalent positions of Lys163 (φ, ψ: -68, 140°) and Ala166 (φ, ψ: -69, 81°) in MJ0391 and MT0146/CbiT, respectively, trigger large deviations in their respective TH elements. Moreover, the β6 & β7 strands in MJ0391 are connected by a type II β-turn. The type II conformation is produced by incorporating Pro165 at the i+1 position. The β-turn is stabilized by an intra-hydrogen bond between the carbonyl-group of Ile164 (i position) and the amino-group of Gly167 (i+3 position). In contrast, in MT0146/CbiT, β6 and β7 are connected by a relatively long loop, comprising six amino acids. The abovementioned structural deviations cause the maximum r.m.s.d value of 6.2 Å between the Cα position of Pro165 in MJ0391 and that of Asp168 in MT0146. The α6 helix also participates in tetrameric interactions (Figure 2c); however, less structural deviation is observed between the corresponding regions of MJ0391 and MT0146/CbiT. In addition, deviations were observed in the non-specific secondary structure regions, such as the N-terminal region and the loops connecting the secondary structural elements (Figure 4a).


Crystal structure of putative CbiT from Methanocaldococcus jannaschii: an intermediate enzyme activity in cobalamin (vitamin B12) biosynthesis.

Padmanabhan B, Yokoyama S, Bessho Y - BMC Struct. Biol. (2013)

Comparison of MJ0391 with MT0146/CbiT. (a) Stereo view of the superposition of MJ0391 (light green) on MT0146/CbiT (red), for all Cα-atoms corresponding to one subunit of the MT0146/CbiT tetramer (PDB ID: 1L3I). (b) The AdoHcy binding site region (stereo view). The interacting residues and the AdoHcy molecule (shown as ‘SAH’ in the figure) of the MT0146/CbiT complex are colored grey & cyan, respectively. The equivalent predicted interacting residues in MJ0391 are yellow. The Tyr65 residue is modeled as Ala, since the electron density corresponding to its side chain was not visible. (c) Electrostatic surface potential of the MJ0391 tetramer. The surfaces are colored blue and red for positive and negative electrostatic surface potentials, denoting less than -15 and greater than +15 KbT, respectively, where Kb is the Boltzmann constant and T is the temperature. The yellow and green arrows indicate the putative precorrin and AdoMet binding pockets, respectively. The surface potential was calculated with the software APBS[27] incorporated in the PyMOL program (Schrödinger, LLC.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Comparison of MJ0391 with MT0146/CbiT. (a) Stereo view of the superposition of MJ0391 (light green) on MT0146/CbiT (red), for all Cα-atoms corresponding to one subunit of the MT0146/CbiT tetramer (PDB ID: 1L3I). (b) The AdoHcy binding site region (stereo view). The interacting residues and the AdoHcy molecule (shown as ‘SAH’ in the figure) of the MT0146/CbiT complex are colored grey & cyan, respectively. The equivalent predicted interacting residues in MJ0391 are yellow. The Tyr65 residue is modeled as Ala, since the electron density corresponding to its side chain was not visible. (c) Electrostatic surface potential of the MJ0391 tetramer. The surfaces are colored blue and red for positive and negative electrostatic surface potentials, denoting less than -15 and greater than +15 KbT, respectively, where Kb is the Boltzmann constant and T is the temperature. The yellow and green arrows indicate the putative precorrin and AdoMet binding pockets, respectively. The surface potential was calculated with the software APBS[27] incorporated in the PyMOL program (Schrödinger, LLC.).
Mentions: Although the overall tertiary structures of the MJ0391 and MT0146/CbiT proteins are essentially similar, striking differences exist in the TH region, an important element for forming the tetrameric arrangements (Figure 4a). The dihedral angles at the equivalent positions of Lys163 (φ, ψ: -68, 140°) and Ala166 (φ, ψ: -69, 81°) in MJ0391 and MT0146/CbiT, respectively, trigger large deviations in their respective TH elements. Moreover, the β6 & β7 strands in MJ0391 are connected by a type II β-turn. The type II conformation is produced by incorporating Pro165 at the i+1 position. The β-turn is stabilized by an intra-hydrogen bond between the carbonyl-group of Ile164 (i position) and the amino-group of Gly167 (i+3 position). In contrast, in MT0146/CbiT, β6 and β7 are connected by a relatively long loop, comprising six amino acids. The abovementioned structural deviations cause the maximum r.m.s.d value of 6.2 Å between the Cα position of Pro165 in MJ0391 and that of Asp168 in MT0146. The α6 helix also participates in tetrameric interactions (Figure 2c); however, less structural deviation is observed between the corresponding regions of MJ0391 and MT0146/CbiT. In addition, deviations were observed in the non-specific secondary structure regions, such as the N-terminal region and the loops connecting the secondary structural elements (Figure 4a).

Bottom Line: The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum.The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity.The putative function of MJ0391 is discussed, based on structural homology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biophysics, National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore 560029, India. paddy@nimhans.kar.nic.in

ABSTRACT

Background: In the anaerobic pathway of cobalamin (vitamin B12) synthesis, the CbiT enzyme plays two roles, as a cobalt-precorrin-7 C15-methyltransferase and a C12-decarboxylase, to produce the intermediate, cobalt-precorrin 8.

Results: The primary structure of the hypothetical protein MJ0391, from Methanocaldococcus jannaschii, suggested that MJ0391 is a putative CbiT. Here, we report the crystal structure of MJ0391, solved by the MAD procedure and refined to final R-factor and R-free values of 19.8 & 27.3%, respectively, at 2.3 Å resolution. The asymmetric unit contains two NCS molecules, and the intact tetramer generated by crystallographic symmetry may be functionally important. The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum.

Conclusions: The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity. The putative function of MJ0391 is discussed, based on structural homology.

Show MeSH
Related in: MedlinePlus