Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids

Mera, Paola E.; Escalante‐Semerena, Jorge C.

doi:10.1074/jbc.m109.059485

Cited by 35 publications

(42 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The calculated molar extinction coefficient for AdoCbl was consistent with values reported in the literature (18). In the Co ϩ assay, cob(II)alamin was chemically reduced to cob(I)alamin using Ti(III)citrate (19), whereas in the Co 2ϩ assay cob(II)alamin was reduced in the active site of EutT by free or protein-bound dihydroflavins as described previously (20). The optimal concentration of free dihydroflavins in the Co 2ϩ assay was found to be 20 M, and reduced flavin adenine dinucleotide (FADH 2 ) was used as the electron donor in the kinetic assays.…”

Section: Construction Of Expression Vectorssupporting

confidence: 71%

“…The assay employed in these experiments provided EutT with cob(II)alamin as the substrate, the cobalamin species that EutT encounters in the cell (20,35). Previously reported kinetic parameters of EutT WT were determined with cob(I)alamin as the substrate; the value for apparent affinity was lower (K m of 4 M) and that for turnover higher (k cat of 0.06 s Ϫ1 ) than the values reported herein (6).…”

Section: Eutt Exhibits Non-michaelis-menten Kinetics Euttmentioning

confidence: 89%

“…Our laboratory has shown that PduO ACAT enzymes can use free dihydroflavins to drive the adenosylation of cob(I-I)alamin (20) and that enzyme activity depended on the generation of a four-coordinate cob(II)alamin species in the active site. The redox potential of free dihydroflavins is not low enough to drive the reduction of free cob(II)alamin to cob(I)alamin in solution, yet it is low enough to reduce the four-coordinate cob(II)alamin bound in the active sites of PduO ACATs.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

2014

Self Cite

View full text Add to dashboard Cite

(adenosylcobalamin, AdoCbl, CoB 12 ) is an essential nutrient for animals, lower eukaryotes, and many prokaryotes. The unique organometallic bond of AdoCbl, between the cobalt ion of the corrinoid and the carbon of the 5=-deoxyadenosyl group, lies at the center of its reactivity. The formation of this bond is catalyzed by ATP:cob(I)alamin adenosyltransferase (ACAT) enzymes (1). There are three types of ACAT enzymes, namely, CobA, PduO, and EutT (2-8). They do not share sequence similarity at the nucleotide level or at the amino acid level, making them a good example of convergent evolution. All three ACAT types were discovered in Salmonella enterica, and they serve distinct growth requirements. The CobA type is the housekeeping ACAT, whereas PduO and EutT are specialized enzymes needed for growth on 1,2-propanediol and ethanolamine, respectively (7, 9).Extensive work has been done to understand the mechanism of corrinoid adenosylation performed by the CobA and PduO type ACATs. These two types of enzymes facilitate the thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin by generating a cob(II)alamin four-coordinate intermediate in the active site of the enzyme (10-12). Both CobA and PduO use conserved aromatic side chains located directly below the cobalt ion to displace the lower ligand of Cbl and generate the four-coordinate species (5, 13). The reduction potential of the cobalt ion in this intermediate is raised enough so that free or protein-bound dihydroflavins can reduce cob(II)alamin to cob(I)alamin (5). Unlike the CobA and PduO type ACAT enzymes, nothing is known about the mechanism of how the thermodynamically unfavorable reduction to cob(II)alamin is facilitated when EutT type ACAT enzymes adenosylate Cbl. In fact, the EutT type enzyme is the least understood of the three types of ACATs because it has not been isolated to homogeneity.We previously reported a method for enriching a sample with EutT using detergents (6). We proposed that the EutT type ACAT enzyme contained a metal cofactor because the protein contains an HX 11 CCXXC 83 motif that resembles those found in Fe/S-containing proteins and inferred that the metal cofactor was labile to oxidation and could be extracted from the protein by metal chelators (6). Problems with the isolation of EutT hindered its biochemical characterization.In this study, we successfully purified wild-type EutT to homogeneity in the absence of detergents. Herein, we report the initial kinetic characterization of EutT and show that the enzyme requires a divalent, transition state metal ion and is most active when in complex with ferrous ions. Results from experiments using homogeneous EutT demonstrate that free dihydroflavins can reduce cob(II)alamin, suggesting that EutT, similar to CobA and PduO, facilitates the unfavorable reduction of cob(II)alamin. This is the first known example of an ACAT requiring an inorganic cofactor for activity. MATERIALS AND METHODS Construction of expression vectors.To generate a recombinant construct of EutT with a...

show abstract

Section: Construction Of Expression Vectorssupporting

confidence: 71%

Section: Eutt Exhibits Non-michaelis-menten Kinetics Euttmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…While the reduction potentials of Co(II)rrinoids in solution (e.g., E °(SHE) = −610 mV for cob(II)alamin [Co(II)Cbl] and −490 mV for cob(II)inamide [Co(II)Cbi + ]) 18 are below those of readily available reducing agents, Co(II)Cbl bound to the PduO-type ACAT from Lactobacillus reuteri ( Lr PduO) and the EutT-type ACAT from S. enterica can be reduced by dihydroflavins (e.g., E °(SHE)= −228 mV for FMN at pH 7.5). 19,20 Although Se CobA-bound Co(II)rrinoids cannot be reduced by free dihydroflavins, they can be converted to the Co(I) state by reduced flavoproteins like flavodoxin. 12,19,21 …”

mentioning

confidence: 99%

Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:Co^Irrinoid Adenosyltransferase Precludes Adenosylation of Incomplete Co^IIrrinoids

et al. 2015

Self Cite

View full text Add to dashboard Cite

Three distinct families of ATP:corrinoid adenosyltransferases (ACATs) exist that are capable of converting vitamin B12 derivatives to coenzyme B12 by catalyzing the thermodynamically challenging reduction of Co(II)rrinoids to form “super-nucleophilic” Co(I) intermediates. While the structures and mechanisms for the other two ACAT families have been studied extensively, little is known about the EutT enzymes beyond the fact that they exhibit a unique requirement for a divalent metal cofactor for enzymatic activity. In this study we have obtained compelling evidence that EutT converts cob(II)alamin to an effectively four-coordinate Co(II) species so as to facilitate Co(II) → Co(I) reduction. Intriguingly, EutT fails to promote axial ligand dissociation for the substrate analogue cob(II)inamide, a natural precursor of cob(II)alamin. This unique substrate specificity of EutT has important physiological implications.

show abstract

“…The lack of metal in Lm EutT is of interest, because in Se EutT Fe(II) somehow disrupts the coordination bond between DMB and the Co(II) ion of the ring. The resulting DMB-off form of the corrinoid substrate results in a Co(II) four-coordinate species whose redox potential is within physiological range and can be readily reduced to Co(I) cobalamin by dihydroflavins 22, 23, 45 . At present, it is unclear how Lm EutT generates the Co(II) four-coordinate species of the cobalamin in the absence of a metal ion.…”

Section: Discussionmentioning

confidence: 99%

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

Costa

Escalante‐Semerena

2018

Biochemistry

Self Cite

View full text Add to dashboard Cite

ATP Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis, and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT (SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-less EutT homologue from Listeria monocytogenes (LmEutT) has ACAT activity, and that unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and displayed positive cooperativity. LmEutT adenosylated cobalamin but not cobinamide, showed specificity for ATP and 2′-deoxyATP, and released a triphosphate by-product. Bioinformatics analyses suggest that metal-less EutT ACATs are also present in other Firmicutes.

show abstract

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids

Abstract: The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive.

Cited by 35 publications

References 40 publications

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:Co^Irrinoid Adenosyltransferase Precludes Adenosylation of Incomplete Co^IIrrinoids

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

Contact Info

Product

Resources

About

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids

Abstract: The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive.

Cited by 35 publications

References 40 publications

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:CoIrrinoid Adenosyltransferase Precludes Adenosylation of Incomplete CoIIrrinoids

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

Contact Info

Product

Resources

About

Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:Co^Irrinoid Adenosyltransferase Precludes Adenosylation of Incomplete Co^IIrrinoids