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Residue Phe112 of the human-type corrinoid adenosyltransferase (PduO) enzyme of Lactobacillus reuteri is critical to the formation of the four-coordinate Co(II) corrinoid substrate and to the activity of the enzyme.

ABSTRACT: ATP:Corrinoid adenosyltransferases (ACAs) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the alpha face of the corrin ring consists of bulky hydrophobic residues. To understand how these residues promote the generation of the four-coordinate cob(II)alamin, variants of the human-type ACA enzyme from L. reuteri (LrPduO) were kinetically and structurally characterized. These studies revealed that residue Phe112 is critical in the displacement of 5,6-dimethylbenzimidazole (DMB) from its coordination bond with the Co ion of the ring, resulting in the formation of the four-coordinate species. An F112A substitution resulted in a 80% drop in the catalytic efficiency of the enzyme. The explanation for this loss of activity was obtained from the crystal structure of the mutant protein, which showed cob(II)alamin bound in the active site with DMB coordinated to the cobalt ion. The crystal structure of an LrPduO(F112H) variant showed a DMB-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency was 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of LrPduO(F112H) suggests that the F112H substitution negatively impacts product release. Substitutions of other hydrophobic residues in the Cbl binding pocket did not result in significant defects in catalytic efficiency in vitro; however, none of the variant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.

SUBMITTER: Mera PE

PROVIDER: S-EPMC2666787 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Residue Phe112 of the human-type corrinoid adenosyltransferase (PduO) enzyme of Lactobacillus reuteri is critical to the formation of the four-coordinate Co(II) corrinoid substrate and to the activity of the enzyme.

Mera Paola E PE St Maurice Martin M Rayment Ivan I Escalante-Semerena Jorge C JC

Biochemistry 20090401 14

ATP:Corrinoid adenosyltransferases (ACAs) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the alpha face of the corrin ring consists of bulky ...[more]

PMID: 19236001

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Project description:The PduO-type adenosine 5'-triphosphate (ATP):corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co(1+)cobalamin (Cbl) and Co(1+)cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi(+)), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co(2+) ? Co(1+) reduction, the enzyme drastically weakens the axial ligand-Co(2+) bond so as to generate effectively four-coordinate (4c) Co(2+)corrinoid species. To explore how LrPduO generates these unusual 4c species, we have used magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopic techniques. The effects of active-site amino acid substitutions on the relative yield of formation of 4c Co(2+)corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an intersubunit salt bridge, and the hydrophobic region surrounding the bound corrin ring. A quantitative analysis of our MCD and EPR spectra indicates that the entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co(2+)corrinoids. Our data also show that a higher level of coordination among several LrPduO amino acid residues is required to exclude the dimethylbenzimidazole moiety of Co(II)Cbl from the active site than to remove the water molecule from Co(II)Cbi(+). Thus, the hydrophilic interactions around and above the corrin ring are more critical to form 4c Co(II)Cbl than 4c Co(II)Cbi(+). Finally, when ATP analogues were used as cosubstrate, only "unactivated" five-coordinate (5c) Co(II)Cbl was observed, disclosing an unexpectedly large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl. Collectively, our results indicate that the level of control exerted by LrPduO over the timing for the formation of the 4c Co(2+)corrinoid intermediates is even more exquisite than previously anticipated.

Dataset Information

Residue Phe112 of the human-type corrinoid adenosyltransferase (PduO) enzyme of Lactobacillus reuteri is critical to the formation of the four-coordinate Co(II) corrinoid substrate and to the activity of the enzyme.

Publications

Residue Phe112 of the human-type corrinoid adenosyltransferase (PduO) enzyme of Lactobacillus reuteri is critical to the formation of the four-coordinate Co(II) corrinoid substrate and to the activity of the enzyme.

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