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 ATP:corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co1+cobalamin (Cbl) and Co1+cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi+), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co2+→Co1+ reduction, the enzyme drastically weakens the axial ligand–Co2+ bond so as to generate effectively four-coordinate (4c) Co2+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 Co2+corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an inter-subunit 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 Co2+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-analogs were used as co-substrate, only “unactivated” 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 Co2+corrinoid intermediates is even more exquisite than previously anticipated.

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri

“…In support of this proposal, we have recently shown that alanine substitutions of active site residues located in the plane of the corrin ring drastically lower the relative yield of four-coordinate Co(II)rrinoid formation achieved by the Lr PduO/ATP complex. 38 …”

“…Yet, it is obvious that non-bonding (electrostatic/hydrophobic) interactions between the corrin ring and the enzyme active site must play an important role in the mechanism of axial ligand removal, as amino acid substitutions in Lr PduO that disrupt the interactions between the peripheral amide groups of the corrin ring and the enzyme active site result in a significantly decreased yield for the five- to four-coordinate Co(II)rrinoid conversion and thus slow down the formation of AdoCbl. 37,38 …”

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation

“…Eº(NHE) = −228 mV for FMN at pH 7.5) 26,27 or by reduced flavoproteins in the case of Se CobA (Eº(NHE) = −440 mV for the semiquinone/reduced flavin couple of FldA, the purported physiological partner of Se CobA). 28–30 Extensive biochemical, 31,32 crystallographic, 32–35 and spectroscopic studies 36–38 of Se CobA and different PduO-type ACATs have revealed that a structurally unique four-coordinate (4c) Co(II)Cbl species is generated by these enzymes in the presence of ATP. The most recent X-ray crystal structures reported for Se CobA and Lr PduO complexed with Co(II)Cbl and MgATP have provided evidence that the DMB moiety that occupies the Coα position of Co(II)Cbl in solution is displaced by a non-coordinating phenylalanine residue (F112 in Lr PduO and F91 in Se CobA).…”

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Evidence of a Tetrahedrally Coordinated Divalent Transition Metal Cofactor with Cysteine Ligation

“…In support of this proposal, it has been shown that amino acid substitutions in the active sites of the CobA- and PduO-type ACATs aimed at altering the relative positioning of the two substrates could cause a significant decrease in catalytic activity, despite the fact that the variants retained the ability to generate sizable amounts of 4c Co(II)Cbl. 16,17 …”

“…11,15 Spectroscopic characterization of enzyme variants revealed that the driving force for the displacement of the DMB ligand is provided by the formation of favorable interactions among active site residues when 4c Co(II)Cbl is generated. 16,17 …”