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Sueharu Horinouchi - One of the best experts on this subject based on the ideXlab platform.
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4 hydroxy 3 methyl 6 1 methyl 2 oxoalkyl pyran 2 one synthesis by a type iii polyketide synthase from rhodospirillum centenum
ChemBioChem, 2013Co-Authors: Takayoshi Awakawa, Sueharu Horinouchi, Yoshinori Sugai, Kanae Otsutomo, Shukun Ren, Shinji Masuda, Yohei Katsuyama, Yasuo OhnishiAbstract:The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl-CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C(10-14) fatty acyl-CoA unit, one malonyl-CoA unit and two Methylmalonyl-CoA units. We identified these products as 4-hydroxy-3-methyl-6-(1-methyl-2-oxoalkyl)pyran-2-ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of Methylmalonyl-CoA as the extender substrate. In addition, in vitro reactions with (13)C-labeled malonyl-CoA revealed that RpsA produced tetraketide 6-alkyl-4-hydroxy-1,5-dimethyl-2-oxocyclohexa-3,5-diene-1-carboxylic acids from C(14-20) fatty acyl-CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.
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the o methyltransferase srsb catalyzes the decarboxylative methylation of alkylresorcylic acid during phenolic lipid biosynthesis by streptomyces griseus
Journal of Bacteriology, 2012Co-Authors: Chiaki Nakano, Nobutaka Funa, Yasuo Ohnishi, Sueharu HorinouchiAbstract:ABSTRACT Streptomyces griseus contains the srs operon, which is required for phenolic lipid biosynthesis. The operon consists of srsA, srsB, and srsC, which encode a type III polyketide synthase, an O-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13′-methyltetradecyl)-4-methylresorcinol, using iso-C16 fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and Methylmalonyl-CoA as extender substrates. An in vitro SrsA reaction using [13C3]malonyl-CoA confirmed that the order of extender substrate condensation was Methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in Streptomyces lividans and used for the in vitro SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of S-adenosyl-l-methionine (SAM). SrsB was incapable of catalyzing the O-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the O-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1). O-methylated alkylresorcylic acid was not detected in the in vitro SrsAB reaction, although it was presumably stable, indicating that O-methylation did not precede decarboxylation. We therefore postulated that O-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.
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phenolic lipids synthesized by type iii polyketide synthase confer penicillin resistance on streptomyces griseus volume 283 2008 pages 13983 13991
Journal of Biological Chemistry, 2008Co-Authors: Masanori Funabashi, Nobutaka Funa, Sueharu HorinouchiAbstract:On Page 13990, the mode of ring folding of the tetraketide intermediate, leading to resorcinol formation, in Fig. 5B was incorrect. The correct ring folding is C-2-C-7 aldol condensation. This error does not change the conclusions of this study. However, the order of condensation of the extender units is updated as Methylmalonyl-CoA, malonyl-CoA, and malonyl-CoA. The corrected figure is shown below. Figure 1
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phenolic lipids synthesized by type iii polyketide synthase confer penicillin resistance on streptomyces griseus
Journal of Biological Chemistry, 2008Co-Authors: Masanori Funabashi, Nobutaka Funa, Sueharu HorinouchiAbstract:Abstract Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria synthesize a variety of aromatic polyketides. A Gram-positive, filamentous bacterium Streptomyces griseus contained an srs operon, in which srsA encoded a type III PKS, srsB encoded a methyltransferase, and srsC encoded a flavoprotein hydroxylase. Consistent with this annotation, overexpression of the srs genes in a heterologous host, Streptomyces lividans, showed that SrsA was a type III PKS responsible for synthesis of phenolic lipids, alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers. In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols from acyl-CoAs of various chain lengths as a starter substrate, one molecule of Methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique in that it incorporated the extender substrates in a strictly controlled order of malonyl-CoA, malonyl-CoA, and Methylmalonyl-CoA to produce alkylresorcinols. An srsA mutant, which produced no phenolic lipids, was highly sensitive to β-lactam antibiotics, such as penicillin G and cephalexin. Together with the fact that the alkylresorcinols were fractionated mainly in the cell wall fraction, this observation suggests that the phenolic lipids, perhaps associated with the cytoplasmic membrane because of their amphiphilic property, affect the characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a variety of bacteria. An srs-like operon is found widely among Gram-positive and -negative bacteria, indicating wide distribution of the phenolic lipids.
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properties and substrate specificity of rppa a chalcone synthase related polyketide synthase in streptomyces griseus
Journal of Biological Chemistry, 2002Co-Authors: Nobutaka Funa, Yutaka Ebizuka, Yasuo Ohnishi, Sueharu HorinouchiAbstract:RppA, a chalcone synthase-related polyketide synthase (type III polyketide synthase) in the bacterium Streptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA. The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined to be 0.93 +/- 0.1 microm and 0.77 +/- 0.04 min(-1), respectively. RppA accepted aliphatic acyl-CoAs with the carbon lengths from C(4) to C(8) as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone synthase-related enzymes found so far in plants and bacteria. RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and Methylmalonyl-CoA as an extender. Thus, the broad substrate specificity of RppA yields a wide variety of products.
Jeremy R Lohman - One of the best experts on this subject based on the ideXlab platform.
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structures of lnmk a bifunctional acyltransferase decarboxylase with substrate analogues reveal the basis for selectivity and stereospecificity
Biochemistry, 2021Co-Authors: Lee M Stunkard, Benjamin J Kick, Jeremy R LohmanAbstract:LnmK stereospecifically accepts (2R)-Methylmalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-Methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and Methylmalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-Methylmalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller malonyl-CoA substrate. Indeed, our kinetic analysis reveals malonyl-CoA is accepted at 1% of the rate of Methylmalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and β-ketoacylsynthases.
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sulfonate nitro bearing methylmalonyl thioester isosteres applied to methylmalonyl coa decarboxylase structure function studies
Journal of the American Chemical Society, 2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of Methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli Methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure-function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases.
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Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to Methylmalonyl-CoA Decarboxylase Structure–Function Studies
2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl-S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure–function relationships. To address this problem, we have synthesized a panel of Methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli Methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure–function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases
Mary E Lidstrom - One of the best experts on this subject based on the ideXlab platform.
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ethylmalonyl coenzyme a mutase operates as a metabolic control point in methylobacterium extorquens am1
Journal of Bacteriology, 2015Co-Authors: Nathan M Good, Cecilia N Martinezgomez, David A C Beck, Mary E LidstromAbstract:The metabolism of one- and two-carbon compounds by the methylotrophic bacterium Methylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-type M. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts for ecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressing ecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation.
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ccrr a tetr family transcriptional regulator activates the transcription of a gene of the ethylmalonyl coenzyme a pathway in methylobacterium extorquens am1
Journal of Bacteriology, 2012Co-Authors: Mary E LidstromAbstract:The ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway is one of the central methylotrophy pathways in Methylobacterium extorquens involved in glyoxylate generation and acetyl-CoA assimilation. Previous studies have elucidated the operation of the ethylmalonyl-CoA pathway in C(1) and C(2) assimilation, but the regulatory mechanisms for the ethylmalonyl-CoA pathway have not been reported. In this study, a TetR-type activator, CcrR, was shown to regulate the expression of crotonyl-CoA reductase/carboxylase, an enzyme of the ethylmalonyl-CoA pathway involved in the assimilation of C(1) and C(2) compounds in Methylobacterium extorquens AM1. A ccrR null mutant strain was impaired in its ability to grow on C(1) and C(2) compounds, correlating with the reduced activity of crotonyl-CoA reductase/carboxylase. Promoter fusion assays demonstrated that the activity of the promoter required for ccr expression (the katA-ccr promoter) decreased as much as 50% in the absence of ccrR compared to wild-type M. extorquens AM1. Gel mobility shift assays confirmed that CcrR directly binds to the region upstream of the katA-ccr promoter. A palindromic sequence upstream of katA at positions -334 to -321 with respect to the predicted translational start site was identified, and mutations in this region eliminated the gel retardation of the katA-ccr promoter region by CcrR. CcrR does not appear to regulate the expression of other ethylmalonyl-CoA pathway genes, suggesting the existence of additional regulators.
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alternative route for glyoxylate consumption during growth on two carbon compounds by methylobacterium extorquens am1
Journal of Bacteriology, 2010Co-Authors: Yoko Okubo, Ludmila Chistoserdova, Song Yang, Mary E LidstromAbstract:Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C(1) and C(2) compounds, showing that both modes of growth have this pathway in common. However, growth on C(2) compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/beta-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C(2) compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and (13)C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/beta-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C(2) compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H(4)F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C(2) compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.
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meab is a component of the methylmalonyl coa mutase complex required for protection of the enzyme from inactivation
Journal of Biological Chemistry, 2004Co-Authors: Natalia Korotkova, Mary E LidstromAbstract:Abstract Adenosylcobalamin-dependent Methylmalonyl-CoA mutase catalyzes the interconversion of Methylmalonyl-CoA and succinyl-CoA. In humans, deficiencies in the mutase lead to methylmalonic aciduria, a rare disease that is fatal in the first year of life. Such inherited deficiencies can result from mutations in the mutase structural gene or from mutations that impair the acquisition of cobalamins. Recently, a human gene of unknown function, MMAA, has been implicated in methylmalonic aciduria (Dobson, C. M., Wai, T., Leclerc, D., Wilson, A., Wu, X., Dore, C., Hudson, T., Rosenblatt, D. S., and Gravel, R. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15554–15559). MMAA orthologs are widespread in bacteria, archaea, and eukaryotes. In Methylobacterium extorquens AM1, a mutant defective in the MMAA homolog meaB was unable to grow on C1 and C2 compounds because of the inability to convert Methylmalonyl-CoA to succinyl-CoA (Korotkova N., Chistoserdova, L., Kuksa, V., and Lidstrom, M. E. (2002) J. Bacteriol. 184, 1750–1758). Here we demonstrate that this defect is not due to the absence of adenosylcobalamin but due to an inactive form of Methylmalonyl-CoA mutase. The presence of active mutase in double mutants defective in MeaB and in the synthesis of either R-Methylmalonyl-CoA or adenosylcobalamin indicates that MeaB is necessary for protection of mutase from inactivation during catalysis. MeaB and Methylmalonyl-CoA mutase from M. extorquens were cloned and purified in their active forms. We demonstrated that MeaB forms a complex with Methylmalonyl-CoA mutase and stimulates in vitro mutase activity. These results support the hypothesis that MeaB functions to protect Methylmalonyl-CoA mutase from irreversible inactivation.
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glyoxylate regeneration pathway in the methylotroph methylobacterium extorquens am1
Journal of Bacteriology, 2002Co-Authors: Natalia Korotkova, Ludmila Chistoserdova, Vladimir Kuksa, Mary E LidstromAbstract:Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that Methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of 14C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and β-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.
Lee M Stunkard - One of the best experts on this subject based on the ideXlab platform.
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structures of lnmk a bifunctional acyltransferase decarboxylase with substrate analogues reveal the basis for selectivity and stereospecificity
Biochemistry, 2021Co-Authors: Lee M Stunkard, Benjamin J Kick, Jeremy R LohmanAbstract:LnmK stereospecifically accepts (2R)-Methylmalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-Methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and Methylmalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-Methylmalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller malonyl-CoA substrate. Indeed, our kinetic analysis reveals malonyl-CoA is accepted at 1% of the rate of Methylmalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and β-ketoacylsynthases.
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sulfonate nitro bearing methylmalonyl thioester isosteres applied to methylmalonyl coa decarboxylase structure function studies
Journal of the American Chemical Society, 2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of Methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli Methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure-function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases.
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Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to Methylmalonyl-CoA Decarboxylase Structure–Function Studies
2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl-S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure–function relationships. To address this problem, we have synthesized a panel of Methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli Methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure–function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases
Peter Dimroth - One of the best experts on this subject based on the ideXlab platform.
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expression of the sodium ion pump methylmalonyl coenzyme a decarboxylase from veillonella parvula and of mutated enzyme specimens in escherichia coli
Journal of Bacteriology, 1995Co-Authors: J B Huder, Peter DimrothAbstract:The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70). A DNA region upstream of mmdA apparently served as promoter in E. coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS(+)-derived expression plasmid. To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter. The derived plasmid, pT7mmd, was transformed into E. coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter. The synthesized proteins showed the typical properties of Methylmalonyl-CoA-decarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin. In Methylmalonyl-CoA-decarboxylase expressed in E. coli from pT7mmd, the gamma subunit was only partially biotinylated and the alpha subunit was present in substoichiometric amounts, resulting in a low catalytic activity. This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed alpha subunit. After these treatments Methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose. To analyze the function of the delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd. E. coli cells transformed with pJHdelta2, which lacks mmdE and the 3' -terminal part of mmdD, showed no Methylmalonyl-CoA-decarboxylase activity. In addition, a contrast, catalytically active Methylmalonyl-CoA-decarboxylase was expressed in E. coli from plasmid pJHdelta1, which contained a deletion of the mmdE gene only. The mutant enzyme could be isolated, reconstituted into proteolipsomes, and shown to function in the transport of Na+ ions coupled to Methylmalonyl-CoA decarboxylation. The small epsilon subunit therefore has no catalytic function within the Methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex.
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sequence of the sodium ion pump methylmalonyl coa decarboxylase from veillonella parvula
Journal of Biological Chemistry, 1993Co-Authors: J B Huder, Peter DimrothAbstract:Abstract The genes encoding Methylmalonyl-CoA decarboxylase from Veillonella parvula were cloned on plasmids using oligonucleotides derived from N-terminal amino acid sequences as specific probes. The entire DNA sequence of the Methylmalonyl-CoA decarboxylase genes together with upstream and downstream regions was determined. The genes encoding subunits alpha (mmdA), delta (mmdD), epsilon (mmdE), gamma (mmdC), and beta (mmdB) of the decarboxylase were clustered on the chromosome in the given order. The previously unnoted epsilon-chain (M(r) 5,888) was clearly shown to be a subunit of the decarboxylase by correspondence of the N-terminal amino acid sequence with that deduced from the DNA sequence of mmdE. The alpha-subunit was 60% identical with the carboxyltransferase domain of rat liver propionyl-CoA carboxylase, the beta-subunit showed 61% sequence identity with the beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae, and the biotin-containing gamma-subunit was 29-39% identical with biotin-domains of other biotin enzymes. The delta-subunit of Methylmalonyl-CoA decarboxylase and the gamma-subunit of oxaloacetate decarboxylase did not show significant sequence homology. The gross structure of both proteins, however, was similar, consisting of a hydrophobic membrane anchor near the N terminus, a proline/alanine linker, and a remarkable accumulation of charged amino acids in the C-terminal part. The sequence of the small epsilon-subunit could be aligned to the C-terminal region of the delta-subunit downstream of the proline/alanine linker, where the two subunits were 47% identical. Of considerable interest for the mechanism of Na+ transport are the long stretches of complete sequence identity between the hydrophobic beta-subunits of Methylmalonyl-CoA decarboxylase and oxaloacetate decarboxylase and the presence of two conserved aspartic acid residues within putative membrane-spanning helices.
