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Papers by Jorge Escalante-Semerena
Journal of the American Chemical Society, Jun 22, 2005
CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo bios... more CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co 1+ corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co 2+/1+ couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co 3+-and Co 2+ corrinoids and the enzyme active site. Our data reveal that while Co 3+ corrinoids interact only weakly with CobA, Co 2+ corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co 2+ corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co 2+ corrinoid species possesses an essentially square-planar Co 2+ center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co 2+ f Co 1+ reduction employed by CobA and Co-C bond-forming enzymes in general are explored.
Scientific Reports, Oct 13, 2022
Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is... more Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5′-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5′-phosphate (α-RP) or α-adenylyl-5′-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT. Cobamides (Cbas) are cobalt-containing cyclic tetrapyrroles belonging to a family of cofactors called 'the pigments of life'. In addition to Cbas, chlorophylls, hemes, and coenzyme F 430 are other metal-containing cofactors that make up this family of biologically important compounds 1. De novo Cba biosynthesis is restricted to some bacteria and archaea, yet Cba-dependent enzymes are found in cells from all domains of life 2,3. The prevalence of Cba-dependent reactions has implicated Cbas as an important compound in communities of organisms 4-6. Cbas are utilized in a diverse array of chemical reactions including enzyme catalyzed carbon skeleton rearrangements, methyl-group transfers and reductive dehalogenation. Additionally, Cbas have been shown to act as photoreceptors in the regulation of carotenoid biosynthesis 7. Cbas are structurally unique from other metal-containing cofactors in that they possess upper (Coβ) and lower (Coα) axial ligands (Fig. 1). Cobalamin (Cbl, B 12) is defined by a 5,6-dimethylbenzimidazole (DMB) lower ligand nucleobase. Adenosylcobalamin (AdoCbl), the coenzyme form of Cbl, is characterized by an upper 5′-deoxyadenosyl moiety which participates in radical chemistry reactions. The lower nucleobase is a source of diversity among Cba structures, incorporating purines, benzimidazoles, and in some cases phenolics 8,9. The Cba that contains adenine as the nucleobase is known as pseudo-cobalamin (psCbl), and it is relevant to this work because psCbl has been reported to be synthesized by methanogenic archaea, and adenine has been shown to be a substrate for MjCobT 10,11. De novo synthesis of Cbas can be divided into early and late steps. The early steps are responsible for the assembly of the corrin ring, while the late steps are responsible for activating and attaching the corrin ring and the nucleobase. The late steps are also referred to as the Nucleotide Loop Assembly (NLA) pathway. In S. enterica, the nicotinate mononucleotide (NaMN):base phosphoribosyltransferase (SeCobT, EC 2.4.2.21) catalyzes the activation
Biochemistry, 2019
The MM2060 (cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein (MmCobD) with L-thr... more The MM2060 (cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein (MmCobD) with L-threonine kinase (PduX) and L-threonine-O-3-phosphate decarboxylase (CobD) activities. In addition to the unexpected L-Thr kinase activity, MmCobD has an extended carboxy-terminal (C-terminal) region annotated as a putative metal-binding zinc finger-like domain. Here we demonstrate that the C-terminus of MmCobD is a ferroprotein containing ~25 non-heme iron atoms per monomer of protein. The absence of the C-terminus substantially reduces, but does not abolish, enzymatic activities in vitro and in vivo. Single residue substitutions of C-terminal putative Fe-binding cysteinyl and histidinyl residues resulted in the loss of Fe and changes in enzyme activity levels. Salmonella enterica ΔpduX or ΔcobD strains were used as heterologous hosts to assess coenzyme B 12 biosynthesis as a function of MmCobD variants. Some of the latter displayed 5-fold higher enzymatic activity in vitro, and enhanced the growth rate of the S. enterica strains that synthesized them. Most of the MmCobD variants tested were 2-to 6-fold less active in vitro, and supported slow growth rates of the S. enterica strains that synthesized them; some substitutions abolished enzyme activity. MmCobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H 2 O 2 and reduction by sodium dithionite. The presence of FeS clusters in MmCobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that MmCobD contains one or more diamagnetic [4Fe-4S] 2+ center(s) that may play a structural or regulatory role.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:
Journal of the American Chemical Society, Jun 22, 2005
CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo bios... more CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co 1+ corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co 2+/1+ couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co 3+-and Co 2+ corrinoids and the enzyme active site. Our data reveal that while Co 3+ corrinoids interact only weakly with CobA, Co 2+ corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co 2+ corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co 2+ corrinoid species possesses an essentially square-planar Co 2+ center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co 2+ f Co 1+ reduction employed by CobA and Co-C bond-forming enzymes in general are explored.
Scientific Reports, Oct 13, 2022
Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is... more Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5′-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5′-phosphate (α-RP) or α-adenylyl-5′-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT. Cobamides (Cbas) are cobalt-containing cyclic tetrapyrroles belonging to a family of cofactors called 'the pigments of life'. In addition to Cbas, chlorophylls, hemes, and coenzyme F 430 are other metal-containing cofactors that make up this family of biologically important compounds 1. De novo Cba biosynthesis is restricted to some bacteria and archaea, yet Cba-dependent enzymes are found in cells from all domains of life 2,3. The prevalence of Cba-dependent reactions has implicated Cbas as an important compound in communities of organisms 4-6. Cbas are utilized in a diverse array of chemical reactions including enzyme catalyzed carbon skeleton rearrangements, methyl-group transfers and reductive dehalogenation. Additionally, Cbas have been shown to act as photoreceptors in the regulation of carotenoid biosynthesis 7. Cbas are structurally unique from other metal-containing cofactors in that they possess upper (Coβ) and lower (Coα) axial ligands (Fig. 1). Cobalamin (Cbl, B 12) is defined by a 5,6-dimethylbenzimidazole (DMB) lower ligand nucleobase. Adenosylcobalamin (AdoCbl), the coenzyme form of Cbl, is characterized by an upper 5′-deoxyadenosyl moiety which participates in radical chemistry reactions. The lower nucleobase is a source of diversity among Cba structures, incorporating purines, benzimidazoles, and in some cases phenolics 8,9. The Cba that contains adenine as the nucleobase is known as pseudo-cobalamin (psCbl), and it is relevant to this work because psCbl has been reported to be synthesized by methanogenic archaea, and adenine has been shown to be a substrate for MjCobT 10,11. De novo synthesis of Cbas can be divided into early and late steps. The early steps are responsible for the assembly of the corrin ring, while the late steps are responsible for activating and attaching the corrin ring and the nucleobase. The late steps are also referred to as the Nucleotide Loop Assembly (NLA) pathway. In S. enterica, the nicotinate mononucleotide (NaMN):base phosphoribosyltransferase (SeCobT, EC 2.4.2.21) catalyzes the activation
Biochemistry, 2019
The MM2060 (cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein (MmCobD) with L-thr... more The MM2060 (cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein (MmCobD) with L-threonine kinase (PduX) and L-threonine-O-3-phosphate decarboxylase (CobD) activities. In addition to the unexpected L-Thr kinase activity, MmCobD has an extended carboxy-terminal (C-terminal) region annotated as a putative metal-binding zinc finger-like domain. Here we demonstrate that the C-terminus of MmCobD is a ferroprotein containing ~25 non-heme iron atoms per monomer of protein. The absence of the C-terminus substantially reduces, but does not abolish, enzymatic activities in vitro and in vivo. Single residue substitutions of C-terminal putative Fe-binding cysteinyl and histidinyl residues resulted in the loss of Fe and changes in enzyme activity levels. Salmonella enterica ΔpduX or ΔcobD strains were used as heterologous hosts to assess coenzyme B 12 biosynthesis as a function of MmCobD variants. Some of the latter displayed 5-fold higher enzymatic activity in vitro, and enhanced the growth rate of the S. enterica strains that synthesized them. Most of the MmCobD variants tested were 2-to 6-fold less active in vitro, and supported slow growth rates of the S. enterica strains that synthesized them; some substitutions abolished enzyme activity. MmCobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H 2 O 2 and reduction by sodium dithionite. The presence of FeS clusters in MmCobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that MmCobD contains one or more diamagnetic [4Fe-4S] 2+ center(s) that may play a structural or regulatory role.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report: