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. 2018 Aug 28;57(34):5088-5095.
doi: 10.1021/acs.biochem.8b00743. Epub 2018 Aug 16.

Spectroscopic Study of the EutT Adenosyltransferase From Listeria Monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate

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Spectroscopic Study of the EutT Adenosyltransferase From Listeria Monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate

Nuru G Stracey et al. Biochemistry. .
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Abstract

The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if this disparity in the metal cofactor requirement among EutT enzymes correlates with differences in substrate specificity or the mechanism of Co(II)Cbl reduction, we employed various spectroscopic techniques to probe the interaction of Co(II)Cbl and cob(II)inamide (Co(II)Cbi+) with LmEutT in the absence and presence of cosubstrate ATP. Our data indicate that LmEutT displays a similar substrate specificity as SeEutT and can bind both Co(II)Cbl and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)Cbl to a 4C species. Notably, LmEutT is the most effective ACAT studied to date in generating the catalytically relevant 4C Co(II)Cbl species, achieving a >98% 5C → 4C conversion yield on the addition of just over one mol equiv of cosubstrate MgATP.

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no competing financial interest

Figures

Figure 1.
Figure 1.
Chemical structure of cob(III)alamin species. In AdoCbl, the variable “upper” axial ligand, X, is Ado as indicated. In cob(III)inamide species the negatively charged nucleotide loop with the DMB base is absent, which causes a water molecule to bind in the “lower” axial position. Upon Co(III)→Co(II) reduction of cob(III)alamin and cob(III)inamide species, the upper axial ligand is lost to generate the five-coordinate, neutral cob(II)alamin [Co(II)Cbl] and mono-cationic cob(II)inamide [Co(II)Cbi+] species.
Figure 2.
Figure 2.
X-ray crystal structure of 4C Co(II)Cbl (black) in the active site of SeCobA, with Mg-ATP located above the corrin ring. Key hydrophobic residues are indicated (pale green) that block off the lower axial position. Note that the DMB base, aminopropanol linkage and nucleotide loop are not resolved in the crystal structure as they are likely solvent exposed.
Figure 3.
Figure 3.
Abs spectra at 4.5 K (gray traces) and variable temperature MCD spectra at 7 T of (A) free Co(II)Cbl, B) Co(II)Cbl in the presence of LmEutT and C) Co(II)Cbl in the presence of LmEutT and 1.3 equiv MgATP. The dashed vertical line indicates the position of the α band.
Figure 4.
Figure 4.
Abs spectra at 4.5 K (gray traces) and variable temperature MCD spectra at 7 T of A) free Co(II)Cbi+, B) Co(II)Cbi+ in the presence of LmEutT C) Co(II)Cbi+ in the presence of LmEutT with 1.3 equiv. MgATP. The vertical lines indicate the position of the α band in the Abs spectra and the shift of the lowest energy feature in the MCD spectra.
Figure 5.
Figure 5.
EPR spectra collected at 20 K of A) free Co(II)Cbl (green), B) Co(II)Cbl incubated with LmEutT (teal), C) Co(II)Cbl in the presence of LmEutT and 1.3 equiv. MgATP (blue) or D) >10 equiv. MgATP (purple), and E) free Co(II)Cbi+ (gray). The simulated spectra (labeled “sim” and shown in lighter colors above experimental traces) were obtained using the fit parameters provided in Table 1.
Figure 6.
Figure 6.
EPR spectra collected at 20 K of A) Co(II)Cbi+ (gray), B) Co(II)Cbi+ in the presence of LmEutT (red), and C) Co(II)Cbi+ in the presence of LmEutT and 10 equiv. MgATP (gold). The simulated spectra (labeled “sim” and shown in lighter colors above experimental traces) were obtained using the fit parameters provided in Table 1.
Figure 7.
Figure 7.
Proposed mechanisms for generation of 4C Co(II)Cbl in the EutT ACATs. In the case of SeEutT (A) Co(II)Cbl can bind to the active site in the absence of ATP as long as the divalent metal cofactor is present (I) to yield a base-off species with an axial water ligand (II). This process is facilitated by the binding of the DMB tail to a specific protein pocket. The subsequent binding of ATP then triggers the formation of 4C Co(II)Cbl (III). For LmEutT (B) that lacks a divalent metal cofactor (I), the enzyme must first bind ATP (II) and then Co(II)Cbl, which is directly converted to a 4C species whereby the DMB base is sequestered in a binding pocket (III).

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