Crystal Structure of Methylmalonyl-Coenzyme A Epimerase from P. shermanii

McCarthy, A.A.; Baker, Heather M.; Shewry, Steven C.; Patchett, Mark L.; Baker, Edward N.

doi:10.1016/s0969-2126(01)00622-0

Cited by 57 publications

(41 citation statements)

References 41 publications

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“…shermanii (NCBI accession number AAL57846) identified only one possible candidate annotated to encode glyoxalase I (NCBI accession number ABA79990), an enzyme of structural and functional homology to methylmalonyl-CoA epimerase (27). The gene called epi (for epimerase) was cloned and expressed, and the protein was heterologously produced as a histidine fusion protein in E. coli (supplemental Fig.…”

Section: Coenzyme B 12 -Dependent Conversion Of Ethylmalonyl-coa Tomentioning

confidence: 99%

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Ethylmalonyl-CoA Mutase from Rhodobacter sphaeroides Defines a New Subclade of Coenzyme B12-dependent Acyl-CoA Mutases

Erb

Rétey²,

Fuchs

et al. 2008

Journal of Biological Chemistry

101

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Coenzyme B 12 -dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B 12 -dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/ methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B 12 -dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.The citric acid cycle has a dual role in central carbon metabolism. On the one hand, it is the terminal respiratory pathway and is used for the complete oxidation of acetyl-CoA to CO 2 . On the other hand, the citric acid cycle is the source of carbon precursors for the synthesis of cell constituents. However, net synthesis of carbon precursors via the citric acid cycle is not possible. Intermediates that are drained from the cycle must be replenished using anaplerotic reaction sequences (1). Therefore, growth on substrates that enter the central carbon metabolism on the level of acetyl-CoA, such as fatty acids, alcohols, and esters but also waxes, alkenes, or C 1 compounds, requires an additional pathway for the assimilation of acetyl-CoA. The glyoxylate cycle that is used for the net conversion of two acetylCoA molecules to malate provides a solution for this problem and has been the only known route for 50 years (2).Recently we proposed the so-called ethylmalonyl-CoA pathway for acetyl-CoA assimilation by Rhodobacter sphaeroides, which lacks isocitrate lyase, the key enzyme of the glyoxylate cycle (3, 4). The new pathway converts three molecules of acetyl-CoA, one molecule of CO 2 , and one molecule of bicarbonate to the citric acid cycle intermediates malate and succinyl-CoA (see Fig. 1). Initially two molecules of acetyl-CoA are converted into crotonyl-CoA involving steps that are common to polyhydroxybutyrate synthesis. Crotonyl-CoA is further transformed in a unique reaction sequence to ␤-methylmalylCoA that is cleaved in the latter part of the pathway to glyoxylate and propionyl-CoA. Glyoxylate condenses with another molecule of acetyl-CoA to form L-malyl-CoA that is in turn hydrolyzed by a so far unknown thioesterase to malate. Propionyl-CoA is assimilated by carboxylation followed by a carbon skeleton rearrangement step catalyzed by coenzyme B 12 -dependent (2R)-methylmalonyl-CoA mutase yielding suc...

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Section: Coenzyme B 12 -Dependent Conversion Of Ethylmalonyl-coa Tomentioning

confidence: 99%

“…Residues that have been implied for Co 2ϩ binding in methylmalonyl-CoA epimerase from P. shermanii are conserved in the enzyme from R. sphaeroides except Gln-65 is replaced by a glutamate residue (27).…”

mentioning

confidence: 99%

Ethylmalonyl-CoA Mutase from Rhodobacter sphaeroides Defines a New Subclade of Coenzyme B12-dependent Acyl-CoA Mutases

Erb

Rétey²,

Fuchs

et al. 2008

Journal of Biological Chemistry

101

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“…The interactions between a dimer are composed mainly of hydrophobic as well as hydrogen bonding interactions. Similarly to GlxI, there are four conserved metal-binding residues, including His 12 , Gln 65 , His 91 , and Glu 141 , in P. shermanii MMCE that locate at the active site (52). The active site geometry of the active MMCE also forms an octahedral coordination with four metalbinding residues and two water molecules around the metal center.…”

Section: Crystal Structure Of Clo Glxi (Inactive Zn 2ϩ -And Active Ni 2ϩmentioning

confidence: 99%

Structural Variation in Bacterial Glyoxalase I Enzymes

Suttisansanee

Lau

Lagishetty

et al. 2011

Journal of Biological Chemistry

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The glyoxalase system catalyzes the conversion of toxic, metabolically produced ␣-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn 2؉ and non-Zn 2؉ activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Grampositive bacteria that has been fully characterized as to its threedimensional structure and its detailed metal specificity. It is a Ni 2؉ /Co 2؉ -activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn 2؉ -bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements. Methylglyoxal (MG),3 an ␣-ketoaldehyde produced from a number of different enzymes and pathways, including triosephosphate isomerase, amino acid degradation, and acetoneconverting monooxygenases, is found in both prokaryotic and eukaryotic organisms (1-4). MG is a cytotoxic compound that can be produced to a level as high as 0.4 mM per cell per day, leading to protein synthesis inhibition, adduct formation with proteins, DNA, and RNA and can promote advanced glycation end products (5-10).A major contributing pathway involved in the detoxification of MG is the glyoxalase system. This two-enzyme system consists of glyoxalase I (GlxI) and glyoxalase II (GlxII) that convert ␣-ketoaldehydes into their corresponding 2-hydroxycarboxylic acids (D-lactate in the case of methylglyoxal), using an intracellular thiol as a cofactor/cosubstrate (Fig. 1). The first enzyme, GlxI (S-D-lactoylglutathione methylglyoxal lyase (isomerizing), EC 4.4.1.5), converts a hemithioacetal, the product of the nonenzymatic reaction between MG and a thiol, such as glutathione (GSH), to S-D-lactoylglutathione. GlxI is a metalloenzyme that can be divided into two classes, Zn 2ϩ activation (i.e. Homo sapiens GlxI (11)) and non-Zn 2ϩ activation (being selectively Ni 2ϩ /Co 2ϩ -activated, i.e. Escherichia coli GlxI (12)). It is likely that almost all Gram-negative prokaryotes possess Ni 2ϩ /Co 2ϩ -activated GlxI with the...

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“…This is due to the observation that the three-dimensional structure of the GlxI enzyme is a repeat of this motif and that there are a number of proteins that have similar folds. Since the fi rst recognition of this fact by Bergdoll, the list of structurally similar folded proteins has grown to include the following: catechol dioxygenases, mitomycin resistance protein, fosfomycin resistance protein (FosA), and methylmalonyl CoA epimerase [113,[126][127][128][129][130].…”

Section: Glyoxalase I As a Member Of The βαβββ Superfamily Of Proteinsmentioning

confidence: 99%

“…This cup-shaped region in βαβββ proteins is functionally conserved as a ligand binding site and also a metal binding site in the case of metalloenzyme representatives of the superfamily. Methylmalonyl CoA epimerase is another metalloenzyme in this superfamily that catalyzes proton transfer using a highly similar active site to GlxI to carry out the R to S conversion of substrate [130]. A crystal structure has been obtained for this enzyme [130], with a metallated active site, but unlike GlxI the active site is contained within one monomer.…”

Section: Glyoxalase I As a Member Of The βαβββ Superfamily Of Proteinsmentioning

confidence: 99%