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.
Highlights
Cobalamin m of and a kcat ofCorrinoid adenosyltransferases play a key role in the biosynthesis of AdoCbl by covalently attaching the 5Ј-deoxyadenosyl moiety from ATP to the Co(I) ion of the corrin ring of Cbl [10, 11]
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
We report the three-dimensional crystal structure of L. reuteri PduO-type corrinoid adenosyltransferase (LrPduO) complexed with MgATP at 1.68-Å resolution and provide a kinetic characterization of the enzyme
Summary
Corrinoid adenosyltransferases play a key role in the biosynthesis of AdoCbl by covalently attaching the 5Ј-deoxyadenosyl moiety from ATP to the Co(I) ion of the corrin ring of Cbl [10, 11] These enzymes generate a biologically unique, labile cobaltcarbon bond that is the source of the unusual chemistry associated with the B12 cofactor. Functional, and structural characterization of PduO-type corrinoid adenosyltransferases has recently been reported, including a high-resolution threedimensional crystal structure of the PduO-type enzyme from the archaeon Thermoplasma acidophilum [23], and two additional homologous structures deposited in the RCSB protein data bank (Bacillus subtilis YvqK, 1RTY; and a putative PduO from Mycobacterium tuberculosis, 2G2D) These structures reveal that the enzyme is a trimer with each subunit composed of a five helix-bundle. The absence of bound substrates in these crystal structures has precluded identification of
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