title: Spectroscopic characterization of active-site variants of the PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri: insights into the mechanism of four-coordinate Co(II)corrinoid formation.
abstract: 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.
authors.affiliation: University of Wisconsin-Madison, Department of Chemistry, Madison, Wisconsin 53706, USA.
authors.forename: Kiyoung
authors.initials: K
authors.lastname: Park
authors.forename: Paola E
authors.initials: PE
authors.lastname: Mera
authors.forename: Jorge C
authors.initials: JC
authors.lastname: Escalante-Semerena
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Brunold
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry*; D045728:Corrinoids / Q000737:chemistry* / Q000378:metabolism; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D008958:Models, Molecular; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D045728:Corrinoids; D003035:Cobalt; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; C082269:ATP-corrinoid adenosyltransferase