title: Kinetic and spectroscopic studies of the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri: substrate specificity and insights into the mechanism of Co(II)corrinoid reduction.
abstract: The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri ( LrPduO) catalyzes the formation of the essential Co-C bond of adenosylcobalamin (coenzyme B 12) by transferring the adenosyl group from cosubstrate ATP to a transient Co (1+)corrinoid species generated in the enzyme active site. While PduO-type enzymes have previously been believed to be capable of adenosylating only Co (1+)cobalamin (Co (1+)Cbl (-)), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co (1+)cobinamide (Co (1+)Cbi) as an alternative substrate. To explore the mechanism by which LrPduO overcomes the thermodynamically challenging reduction of its Co (2+)corrinoid substrates, we have examined how the enzyme active site alters the geometric and electronic properties of Co (2+)Cbl and Co (2+)Cbi (+) by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic techniques. Our data reveal that upon binding to LrPduO that was preincubated with ATP, both Co (2+)corrinoids undergo a partial ( approximately 40-50%) conversion to distinct paramagnetic Co (2+) species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co (2+) complexes, based on a comparison with the results obtained in our previous studies of related enzymes. Consequently, it appears that the general strategy employed by adenosyltransferases for effecting Co (2+) --> Co (1+) reduction involves the formation of an "activated" Co (2+)corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3d z (2) -based molecular orbital.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, 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* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism; D045728:Corrinoids / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D003038:Cobamides; D045728:Corrinoids; D003035:Cobalt; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; C082269:ATP-corrinoid adenosyltransferase; C000913:cobamamide