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• W1989044022 abstract "Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional 1H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime.Background: 2-Hydroxy-3-oxoadipate synthase is predicted to be essential for growth of Mycobacterium tuberculosis and is subject to allosteric regulation.Results: The role of regulators was characterized by kinetics and detection of thiamin diphosphate-bound covalent intermediate distribution.Conclusion: AcCoA activates steps leading to a predecarboxylation intermediate; GarA chiefly inhibits postdecarboxylation steps.Significance: This novel regulatory regime in thiamin diphosphate enzymes provides new leads to inhibition. Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional 1H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime. Background: 2-Hydroxy-3-oxoadipate synthase is predicted to be essential for growth of Mycobacterium tuberculosis and is subject to allosteric regulation. Results: The role of regulators was characterized by kinetics and detection of thiamin diphosphate-bound covalent intermediate distribution. Conclusion: AcCoA activates steps leading to a predecarboxylation intermediate; GarA chiefly inhibits postdecarboxylation steps. Significance: This novel regulatory regime in thiamin diphosphate enzymes provides new leads to inhibition. Thiamin diphosphate (ThDP) 2The abbreviations used are: ThDPthiamin diphosphateAcCoAacetyl coenzyme AHOAS2-hydroxy-3-oxoadipate synthase (wild type cloned from M. tuberculosis and overexpressed in E. coli)HOA2-hydroxy-3-oxoadipateMSPmethyl succinyl phosphonateMSP-ThDPadduct of MSP with ThDP at the C2 positionenaminepostdecarboxylation intermediate from the predecarboxylation adduct of 2-ketoglutarate with ThDP at the C2 positionHBThDPC2-hydroxybutanoate-ThDPHOA-ThDPadduct of HOA with ThDP at the C2 positionE1oE1 component of the 2-ketoglutarate dehydrogenase complexBis-Trisbis(2-hydroxyethyl)aminotris(hydroxymethyl)methaneSUMOsmall ubiquitin-like modifier2KG2-ketoglutarate. -dependent 2-ketoglutarate:glyoxylate carboligase activity and the resultant production of 2-hydroxy-3-oxoadipate (HOA) reported in both bacterial and mammalian cell extracts (1.O'Fallon J.V. Brosemer R.W. Cellular localization of α-ketoglutarate:glyoxylate carboligase in rat tissues.Biochim. Biophys. Acta. 1977; 499: 321-328Crossref PubMed Scopus (18) Google Scholar, 2.Yamasaki H. Moriyama T. Purification, general properties and two other catalytic activities of α-ketoglutarate:glyoxylate carboligase of Mycobacterium phlei.Biochim. Biophys. Acta. 1971; 242: 637-647Crossref PubMed Scopus (4) Google Scholar, 3.Yamasaki H. Moriyama T. α-Ketoglutarate:glyoxylate carboligase activity in Escherichia coli.Biochem. Biophys. Res. Commun. 1970; 39: 790-795Crossref PubMed Scopus (10) Google Scholar, 4.Yamasaki H. Moriyama T. Inhibitory effect of α-ketoglutarate:glyoxylate carboligase activity on porphyrin synthesis in mycobacterium phlei.Biochem. Biophys. Res. Commun. 1970; 38: 638-643Crossref PubMed Scopus (10) Google Scholar, 5.Schlossberg M.A. Richert D.A. Bloom R.J. Westerfeld W.W. Isolation and identification of 5-hydroxy-4-ketovaleric acid as a product of α-ketoglutarate:glyoxylate carboligase.Biochemistry. 1968; 7: 333-337Crossref PubMed Scopus (21) Google Scholar) are assigned to the E1 component of the 2-ketoglutarate dehydrogenase complex (E1o) based on experiments with the purified enzymes in vitro (see Scheme 1). In mycobacterial cell extracts, the 2-ketoglutarate dehydrogenase complex activity could not be detected, and the E2o component could not be identified (6.Tian J. Bryk R. Itoh M. Suematsu M. Nathan C. Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of α-ketoglutarate decarboxylase.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10670-10675Crossref PubMed Scopus (158) Google Scholar). However, the product of the gene Rv1248c (annotated as the E1o of Mycobacterium tuberculosis 2-ketoglutarate dehydrogenase complex) could catalyze the synthesis of HOA in vitro using either 2-ketoglutarate and glyoxylate or a small molecule extract of mycobacteria as a source of substrates. Overexpression of Rv1248c in M. tuberculosis led to an increase in the abundance of HOA (detected as 2-hydroxylevulinate by mass spectrometry), suggesting that the HOA synthase activity arises from Rv1248c in vivo (7.de Carvalho L.P. Zhao H. Dickinson C.E. Arango N.M. Lima C.D. Fischer S.M. Ouerfelli O. Nathan C. Rhee K.Y. Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.Chem. Biol. 2010; 17: 323-332Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The metabolic fate of HOA is unknown. HOA synthase activity has been implicated in glyoxylate detoxification in mouse liver (8.Bunik V.I. Fernie A.R. Metabolic control exerted by the 2-oxoglutarate dehydrogenase reaction: a cross-kingdom comparison of the crossroad between energy production and nitrogen assimilation.Biochem. J. 2009; 422: 405-421Crossref PubMed Scopus (104) Google Scholar). thiamin diphosphate acetyl coenzyme A 2-hydroxy-3-oxoadipate synthase (wild type cloned from M. tuberculosis and overexpressed in E. coli) 2-hydroxy-3-oxoadipate methyl succinyl phosphonate adduct of MSP with ThDP at the C2 position postdecarboxylation intermediate from the predecarboxylation adduct of 2-ketoglutarate with ThDP at the C2 position C2-hydroxybutanoate-ThDP adduct of HOA with ThDP at the C2 position E1 component of the 2-ketoglutarate dehydrogenase complex bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane small ubiquitin-like modifier 2-ketoglutarate. Acetyl coenzyme A (AcCoA) was recently identified as an allosteric activator, and GarA (Rv1827) was identified as an allosteric inhibitor of Mycobacterium smegmatis 2-ketoglutarate decarboxylase, a homolog of HOAS (9.Wagner T. Bellinzoni M. Wehenkel A. O'Hare H.M. Alzari P.M. Functional plasticity and allosteric regulation of α-ketoglutarate decarboxylase in central mycobacterial metabolism.Chem. Biol. 2011; 18: 1011-1020Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), with respect to both its 2-ketoglutarate decarboxylase activity and its HOA synthase activity. However, GarA inhibited the two activities to a different extent. To gain more insight into the function of this still enigmatic enzyme, we attempted to identify the individual chemical steps affected by these allosteric modulators. We used circular dichroism (CD) spectroscopy to monitor accumulation of a stable predecarboxylation intermediate analog and chemical quench NMR to assess rate-limiting steps to gain further insight into the various reactions catalyzed by HOAS under multiple conditions. These methods allowed us to study events in active sites containing ThDP so that the effect of allosteric regulation on individual catalytic steps could be determined. Alcohol dehydrogenase from horse liver, β-mercaptoethanol, β-NADH, Bis-Tris, Na2EDTA, acetyl coenzyme A, potassium phosphate, disodium 2-ketoglutarate, glyoxylate, MES, phenylmethanesulfonyl fluoride (PMSF), potassium ferricyanide, carbenicillin, kanamycin, MgCl2, thiamin hydrochloride, and thiamin diphosphate were obtained from Sigma. Dithiothreitol (DTT) was from USB Corp. (Cleveland, OH), and isopropyl β-d-1-thiogalactopyranoside was from Denville Scientific (Metuchen, NJ). Methyl succinyl phosphonate (MSP) disodium salt was synthesized as reported (10.Shim da J. Nemeria N.S. Balakrishnan A. Patel H. Song J. Wang J. Jordan F. Farinas E.T. Assignment of function to histidines 260 and 298 by engineering the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions that lead to acceptance of substrates lacking the 5-carboxyl group.Biochemistry. 2011; 50: 7705-7709Crossref PubMed Scopus (20) Google Scholar). The synthesis of [C2,C6′-13C2]ThDP has been reported (11.Balakrishnan A. Nemeria N.S. Chakraborty S. Kakalis L. Jordan F. Determination of pre-steady-state rate constants on the Escherichia coli pyruvate dehydrogenase complex reveals that loop movement controls the rate-limiting step.J. Am. Chem. Soc. 2012; 134: 18644-18655Crossref PubMed Scopus (30) Google Scholar). Endogenous HOAS from M. tuberculosis lysates was immunoprecipitated as described earlier (7.de Carvalho L.P. Zhao H. Dickinson C.E. Arango N.M. Lima C.D. Fischer S.M. Ouerfelli O. Nathan C. Rhee K.Y. Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.Chem. Biol. 2010; 17: 323-332Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The samples were subjected to SDS-PAGE (7.5%), and proteins migrating at the Mr ∼135,000 band were subjected to tryptic digestion followed by LC/MS analysis to detect enrichment of HOAS. In a duplicate experiment, after SDS-PAGE (7.5%) separation, the proteins were transferred to a PVDF membrane, and the HOAS-enriched band was subjected to Edman degradation for N-terminal sequence determination. The M. tuberculosis ketoglutarate decarboxylase (kgd) gene (Rv1248c) was PCR-amplified from H37Rv genomic DNA using the forward primer 5′-TCGAGGCGAACAGCGCATATGGCCAACATAA-3′ and reverse primer 5′-ATGTCTCCTCGAGCGTTAAGCTTAGCGAA-3′. Restriction sites for NdeI and HindIII, respectively, are underlined. The PCR fragment was cloned into the pET-11c vector, and the resulting plasmid was transformed into Escherichia coli BL21(DE3) CodonPlus-RILP competent cells. Cultures were grown in LB medium containing 50 mg/liter carbenicillin, 1 mm thiamin, and 1 mm MgCl2 at 37 °C with shaking at 220 rpm. At an A600 of ∼0.6–0.8, target protein overexpression was induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside, and the culture was incubated at 18 °C while shaking at 220 rpm overnight. Harvested cells were washed with PBS and resuspended in 40 ml of 20 mm KH2PO4 (pH 7.4) containing 0.2 mm ThDP and 5 mm MgCl2 (Buffer A). PMSF (1 mm final concentration), lysozyme (0.6 mg/ml final concentration), one protease inhibitor mixture tablet, and 1 μg/ml DNase were added to the cell suspension and incubated at 37 °C for 30 min. The cell suspension was passed twice through a French press operating at 1000 p.s.i. in an ice-cooled chamber. All further operations were performed at 4 °C unless otherwise noted. The lysates were clarified by centrifugation at 35,000 × g for 25 min. The supernatant was diluted to 200 ml in Buffer A, and solid (NH4)2SO4 was added to make the solution 15% (w/v) in (NH4)2SO4 with stirring for 30 min. The resulting suspension was centrifuged at 35,000 × g for 25 min, and solid (NH4)2SO4 was added to the supernatant to make the solution 45% (w/v) saturated with stirring for 30 min. The resulting clear white suspension was centrifuged at 35,000 × g for 25 min, and the pellets were collected, dissolved in Buffer A, and dialyzed against 4 liters of Buffer A overnight. Batch purification was performed using a Q Sepharose FF column (20 ml). The column was washed with Buffer A, and bound proteins were eluted with Buffer B (Buffer A + 1 m NaCl) with a linear gradient in 15 column volumes. Fractions containing HOAS (as determined by SDS-PAGE) were pooled, concentrated, and dialyzed against Buffer A overnight. Additional purification was achieved by using a Mono Q (8-ml) column equilibrated with Buffer A and eluted using Buffer B with a linear gradient in 11 column volumes. Fractions containing HOAS were pooled and purified further on Sephacryl S-100 HR (320 ml) equilibrated with Buffer A and eluted with Buffer A. Fractions containing HOAS were pooled, concentrated, flash frozen in liquid N2, and stored at −80 °C until use. In a typical batch, protein was purified to ∼90% homogeneity according to Coomassie staining and densitometry analysis. The N-terminal sequence of a sample of the purified recombinant protein was confirmed by Edman degradation. The M. tuberculosis garA gene (Rv1827) was PCR-amplified from H37Rv DNA using the forward primer 5′-TCAGTGACGGACATGGAACCCGG-3′ and the reverse primer 5′-TCACGGGCCCCCGGTACT-3′. The PCR fragment containing additional TCA coding for N-terminal Ser (to assist in complete Ulp protease cleavage of the SUMO tag) was cloned into the pET-SUMO vector using the TA Cloning® method. After the sequence had been verified, the resulting plasmid was transformed into E. coli BL21(DE3) CodonPlus-RILP competent cells. Three liters of LB containing 50 mg/liter kanamycin were inoculated with 60 ml of overnight starter culture and incubated at 37 °C with shaking at 220 rpm. At an A600 of ∼0.6–0.8, target protein overexpression was induced with 1.0 mm isopropyl β-d-1-thiogalactopyranoside, and the culture was incubated at 37 °C while shaking at 220 rpm for 6 h. The harvested cells were washed with PBS and resuspended in 40 ml of 25 mm Tris (pH 8.0) (Buffer C). PMSF (1 mm final concentration), lysozyme (0.6 mg/ml final concentration), one protease inhibitor mixture tablet, and 1 μg/ml DNase were added to the suspension and incubated in a 37 °C water bath for 30 min. The lysozyme-treated cell suspension was passed twice through a French press operating at 1000 p.s.i. in an ice-cooled chamber. All further operations were performed at 4 °C unless otherwise noted. The lysed cells were clarified by centrifugation at 35,000 × g for 25 min. Batch purification was performed with a His-Trap FF (5 ml) column washed with Buffer C, and the protein was eluted with Buffer D (Buffer C + 500 mm imidazole) with a linear gradient in 7 column volumes. Fractions containing the target SUMO fusion protein were pooled and treated with ubiquitin-like protein 1 (Invitrogen) at 1 μg/mg protein and incubated at ambient temperature for 6 h. Next, the mixture was loaded onto a His-Trap FF column (5 ml) equilibrated with Buffer C and eluted with Buffer D. The flow-through containing GarA without the fusion SUMO tag was concentrated and dialyzed against Buffer C overnight. Aliquots of the dialyzed protein were flash frozen in liquid N2 and stored at −80 °C until use. Protein concentrations were determined using the Bradford assay method (12.Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215560) Google Scholar). Ferricyanide reductase assays were performed as described (6.Tian J. Bryk R. Itoh M. Suematsu M. Nathan C. Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of α-ketoglutarate decarboxylase.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10670-10675Crossref PubMed Scopus (158) Google Scholar). Succinate semialdehyde production was monitored using horse liver alcohol dehydrogenase in the presence of NADH, and the disappearance of NADH was monitored at 340 nm. The activity was measured in the presence of 20 mm 2-ketoglutarate in a 1-ml volume of standard assay buffer (20 mm Bis-Tris, 1 mm ThDP, 5 mm MgCl2, 0.2 mg/ml NADH, 0.08 mg/ml alcohol dehydrogenase (pH 6.5)) at 37 °C. The reaction was started with addition of 1 μm HOAS. Assays were performed using a Uvikon XL UV-visible spectrophotometer (SI Analytics GmbH, Mainz, Germany) equipped with a circulating water bath and thermospacers operating at 37 °C. Assays were performed in triplicate, and the experiment was performed at least twice. A three-component buffer system (50 mm acetic acid, 50 mm MES, and 100 mm Tris) (13.Ellis K.J. Morrison J.F. Buffers of constant ionic strength for studying pH-dependent processes.Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (648) Google Scholar) containing additional 500 μm ThDP and 5 mm MgCl2 was used in the pH range 4.5–8.5 to assay for the ferricyanide reductase and carboligase activities. The pH dependence of the kinetic parameters for the ferricyanide reductase activity was determined using the assay modified for the plate reader format described in the following section. CD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics, Leatherhead, UK) in a 1-cm-path length cell in the near-UV (280–400-nm) wavelength region. Time-dependent synthesis of chiral 2-hydroxy-3-oxoadipate by HOAS from 2-ketoglutarate and glyoxylate was monitored continuously at 278 nm in the kinetics mode by CD. A typical reaction mixture in a 2.4-ml cuvette contained 20 mm Bis-Tris (pH 6.5), 200 μm ThDP, 5 mm MgCl2, and varying concentrations of either 2-ketoglutarate or glyoxylate in the presence of a fixed (10 mm) concentration of the second substrate. The reaction was started by addition of 500 nm HOAS and was monitored for 500 s at 37 °C, and data points were averaged every 10 s. Lineweaver-Burk plots were obtained by varying 2-ketoglutarate (0.5 1.0, 2.0, 5.0, and 10.0 mm) in the presence of various fixed concentrations of glyoxylate (0.5, 1.0, 2.0, 5.0, and 10.0 mm). Steady state velocities calculated from the linear region of the progress curves were either fit to a hyperbolic Michaelis-Menten plot (Equation 1) or to Equation 2 when linear substrate inhibition at high concentrations was observed. (v/E)=(Vmax/E)×SKm+S(Eq. 1) (v/E)=(Vmax/E)×SKm+S(1+S/Ki)(Eq. 2) In this expression, v is the steady state linear velocity, Vmax is the maximal velocity at saturating substrate, E is the enzyme concentration, S is the concentration of the varied substrate, Km is the Michaelis constant for the varied substrate, and Ki is the apparent substrate inhibition constant at high substrate concentration. A typical ferricyanide reductase assay reaction mixture (200 μl) in 20 mm Bis-Tris (pH 6.5) contained 5 mm MgCl2, 1.6 mm K3Fe(CN)6, 200–1000 nm HOAS, and varying amounts of ThDP (0–500 μm), 2-ketoglutarate (0–20 mm), AcCoA (0–2000 μm), MSP (0–2000 μm), or GarA (0–30 μm). The time-dependent decrease in absorbance at 420 nm was monitored over 20 min at 37 °C. The linear region of the progress curves was used to calculate the steady state velocities. All assays were carried out in triplicate in Corning 96-well transparent clear bottom plates using a Spectramax M5 plate reader (Molecular Devices, Sunnyvale, CA). Kinetic data were fitted using the nonlinear, least square, curve fitting program of SigmaPlot v.10.0 for Windows. The data points are the means of experimental triplicates, and the error bars are the standard deviation. The solid lines are the regression fits. Standard errors are reported with the fitted constants. The kinetic parameters kcat, Km, and kcat/Km obtained from Michaelis-Menten plots upon varying either 2-ketoglutarate or ThDP in the presence of various fixed concentrations of the allosteric regulators were fit to Equation 3 for hyperbolic increase, Equation 4 for hyperbolic increase with reversal at high concentrations, or Equation 5 for hyperbolic decrease. β=β0+βmax×[L]KL+[L](Eq. 3) β=β0+βmax×[L]KL+[L]×(1+[L]/KR)(Eq. 4) β=β0−(β0−βmin)×[L]nKLn+[L]n(Eq. 5) In these expressions, L is the concentration of the varied regulator, β is the value of the kinetic parameters at various L, β0 is the value in the absence of the regulator, βmax is the maximal value, and βmin is the minimal value. KL is the concentration of the regulator at which the half-maximal regulatory effect is observed on each of the parameters. KR is the constant for loss of activation, and n is the Hill coefficient. Ki of MSP was estimated by global fit of inhibition performed using Equation 6 where the varied substrate S is 2-ketoglutarate. (v/E)=(Vmax/E)×SS+Km(1+[MSP]/Ki)(Eq. 6) The effect of regulators on the steady state linear velocity in the (R)-HOA synthase assay was determined by fitting the data to Equation 3 for AcCoA and Equation 5 for GarA. Here, β is the steady state linear velocity v/E where E is the enzyme concentration. L is the concentration of the regulator, and KL is the apparent activation or inactivation constant. Data for GarA, plotted in the fractional inhibition form, were fitted to Equation 3. Here, β is the percentage of inhibition, β0 was set to zero, and βmax is the maximal percentage of inhibition. L is the concentration of GarA, and KL is the apparent inactivation constant, Kinact. Ki of GarA was estimated by global fit of inhibition performed using Equation 7 where the varied substrate S is glyoxylate. All data were fit by nonlinear regression using SigmaPlot v.10.0. (v/E)=(Vmax/E)×S(S+Km)×(1+[GarA]/Ki)(Eq. 7) To a 2.4-ml cuvette containing HOAS (2.75 mg/ml; 20 μm) in 20 mm KH2PO4 (pH 7.0) with additional 200 μm ThDP and 5 mm MgCl2, incremental amounts of MSP were added from a 1 m stock solution to obtain the desired final concentrations (0–20 mm). After mixing for 10 s and incubation for an additional 30 s, near-UV (280–400 nm) CD spectra were collected at 25 °C. The procedure was essentially the same for experiments with HOAS (20 μm) in the presence of saturating concentrations of the allosteric regulator, either AcCoA (500 μm) or GarA (50 μm). For quantitative analysis, the amplitude of the CD band pertaining to the 1′,4′-iminopyrimidine tautomer of the MSP-ThDP complex at 302 nm corrected for dilution was plotted against MSP concentration. Apparent dissociation constants (Kdapp) were calculated by fitting the data to the Hill function described in Equation 8 using SigmaPlot v.10.0. CD302=CD0+CD302max×[MSP]n(Kdapp)n+[MSP]n(Eq. 8) In this expression, CD302 is the observed CD signal at the given wavelength, CD0 is the CD signal of the protein at this wavelength in the absence of MSP, CD302max is the maximum CD signal at saturation with MSP, [MSP] is the concentration of substrate analog, and n is the Hill coefficient. Apo-HOAS (25 mg/ml; 185.2 μm) was incubated with 2 eq of [C2,C6′-13C2]ThDP (370.4 μm) in 20 mm KH2PO4 buffer (pH 7.0) containing MgCl2 (5 mm) at 4 °C for 30 min. For experiments with the allosteric modulators, the enzyme reconstituted with labeled ThDP was divided into 200-μl aliquots, 2 eq of GarA (370.4 μm) or AcCoA (370.4 μm) were added, and the mixture was incubated for 30 min on ice. Typical reactions were started by addition of substrates to a final concentration of 40 mm each to 200 μl of the enzyme aliquot at 25 °C, and the reaction was stopped after 30 s by addition of 100 μl of 12.5% TCA in 1 m DCl/D2O. The carboligase reaction in the presence of activator AcCoA was stopped at 5 s. The quenched mixtures were centrifuged at 15,700 × g for 30 min, and the supernatant was filtered using a Gelman nylon Acrodisc (0.45 μm). D2O (80 μl) was added to the filtered samples. The resultant samples were analyzed by a one-dimensional 1H gradient carbon heteronuclear single quantum coherence experiment on a Varian 600-MHz NMR instrument. Data acquisition was at 25 °C, and sample pH was ∼0.75. 2816 transients in the 1H dimension were collected with a recycle delay time of 2 s. Data processing (exponential window function and zero filling) and analyses (base-line correction, integration, and signal-to-noise ratio estimates) were performed using ACD/NMR processor academic edition (v12.01, Advanced Chemistry Development, Inc., Toronto, Canada). The relative integrals of C6′-H signals of respective ThDP-bound covalent intermediates represent their relative ratios after correction for the amount of unbound ThDP estimated using Equations 9 and 10. [E−ThDP]=[ThDP]0+[E]0+Kd2 −([ThDP]0+[E]0+Kd)24−[ThDP]0×[E]0(Eq. 9) [ThDP]=[ThDP]0−[E−ThDP](Eq. 10) Here, [E-ThDP] represents the concentration of HOAS-ThDP complex, [ThDP] and [ThDP]0 represent concentrations of free and total ThDP, and Kd is the dissociation constant. The bioinformatics prediction of the N terminus of HOAS has been revised since the M. tuberculosis enzyme was first studied (14.de Souza G.A. Målen H. Søfteland T. Saelensminde G. Prasad S. Jonassen I. Wiker H.G. High accuracy mass spectrometry analysis as a tool to verify and improve gene annotation using Mycobacterium tuberculosis as an example.BMC Genomics. 2008; 9: 316Crossref PubMed Scopus (61) Google Scholar). To ensure that our recombinant protein included the native N terminus, we identified the N-terminal start sequence of endogenous HOAS. We immunoprecipitated HOAS from M. tuberculosis H37Rv lysates using α-HOAS antibodies (7.de Carvalho L.P. Zhao H. Dickinson C.E. Arango N.M. Lima C.D. Fischer S.M. Ouerfelli O. Nathan C. Rhee K.Y. Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.Chem. Biol. 2010; 17: 323-332Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and subjected the band corresponding to the target protein to Edman sequencing. The first 5 amino acids were determined to be ANISS in agreement with recent observations (15.Kelkar D.S. Kumar D. Kumar P. Balakrishnan L. Muthusamy B. Yadav A.K. Shrivastava P. Marimuthu A. Anand S. Sundaram H. Kingsbury R. Harsha H.C. Nair B. Prasad T.S. Chauhan D.S. Katoch K. Katoch V.M. Chaerkady R. Ramachandran S. Dash D. Pandey A. Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Google Scholar). Next, HOAS was cloned into the pET-11c vector, and tagless recombinant HOAS was overexpressed and purified to >90% purity as judged by SDS-PAGE. The N-terminal start sequence of the recombinant enzyme was confirmed by Edman sequencing. GarA was PCR-amplified and cloned into the pET-SUMO vector, and recombinant GarA was overexpressed and purified to >95% purity. Approximately 15 mg of pure HOAS and 15 mg of pure GarA were obtained per liter of culture. Carboligase products of ThDP-dependent enzymes can be detected by CD spectroscopy in the near-UV region (250–400 nm) because the products are chiral. A negative band at λmax = 278 nm showed a time-dependent increase in the presence of HOAS (500 nm), 2-ketoglutarate, and glyoxylate (Fig. 1A). This band, suggestive of accumulation of a chiral product, was observed in the supernatant after enzyme removal and did not appear when one of the three components was omitted. Earlier, 1H NMR and LC-MS analyses of this reaction assigned the product as 2-hydroxy-3-oxoadipate, which is decarboxylated non-enzymatically to hydroxylevulinate (7.de Carvalho L.P. Zhao H. Dickinson C.E. Arango N.M. Lima C.D. Fischer S.M. Ouerfelli O. Nathan C. Rhee K.Y. Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.Chem. Biol. 2010; 17: 323-332Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). By analogy with carboligase reactions of other ThDP-dependent enzymes (16.Baykal A. Chakraborty S. Dodoo A. Jordan F. Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent d" @default.
• W1989044022 created "2016-06-24" @default.
• W1989044022 creator A5000793262 @default.
• W1989044022 creator A5009378425 @default.
• W1989044022 creator A5044356220 @default.
• W1989044022 date "2013-07-01" @default.
• W1989044022 modified "2023-10-17" @default.
• W1989044022 title "Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase" @default.
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