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Structural characterization of the active site of the PduO-type ATP:Co(I)rrinoid adenosyltransferase from Lactobacillus reuteri.

ABSTRACT: The three-dimensional crystal structure of the PduO-type corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) has been solved to 1.68-A resolution. The functional assignment of LrPduO as a corrinoid adenosyltransferase was confirmed by in vivo and in vitro evidence. The enzyme has an apparent Km(ATP) of 2.2 microM and Km(Cobalamin) of 0.13 microM and a kcat of 0.025 s(-1). Co-crystallization of the enzyme with Mg-ATP resulted in well-defined electron density for an N-terminal loop that had been disordered in other PduO-type enzyme structures. This newly defined N-terminal loop makes up the lower portion of the enzyme active site with the other half being contributed from an adjacent subunit. These results provide the first detailed description of the enzyme active site for a PduO-type adenosyltransferase and identify a unique ATP binding motif at the protein N terminus. The molecular architecture at the active site offers valuable new insight into the role of various residues responsible for the human disease methylmalonic aciduria.

SUBMITTER: St Maurice M

PROVIDER: S-EPMC6574208 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Structural characterization of the active site of the PduO-type ATP:Co(I)rrinoid adenosyltransferase from Lactobacillus reuteri.

St Maurice Martin M Mera Paola E PE Taranto María P MP Sesma Fernando F Escalante-Semerena Jorge C JC Rayment Ivan I

The Journal of biological chemistry 20061122 4

The three-dimensional crystal structure of the PduO-type corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) has been solved to 1.68-A resolution. The functional assignment of LrPduO as a corrinoid adenosyltransferase was confirmed by in vivo and in vitro evidence. The enzyme has an apparent Km(ATP) of 2.2 microM and Km(Cobalamin) of 0.13 microM and a kcat of 0.025 s(-1). Co-crystallization of the enzyme with Mg-ATP resulted in well-defined electron density for an N-terminal loop t ...[more]

PMID: 17121823

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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

Structural characterization of the active site of the PduO-type ATP:Co(I)rrinoid adenosyltransferase from Lactobacillus reuteri.

Publications

Structural characterization of the active site of the PduO-type ATP:Co(I)rrinoid adenosyltransferase from Lactobacillus reuteri.

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