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 (1)H 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.
Highlights
2-hydroxy-3-oxoadipate (HOA) reported in both bacterial and mammalian cell extracts [1,2,3,4,5] are assigned to the E1 component of the 2-ketoglutarate dehydrogenase complex (E1o) based on experiments with the purified enzymes in vitro
Overexpression of Rv1248c in M. tuberculosis led to an increase in the abundance of HOA, suggesting that the HOA synthase activity arises from Rv1248c in vivo [7]
All three pathways are derived from thiamin diphosphate (ThDP)-catalyzed decarboxylation of 2-ketoglutarate and the reaction of the enamine intermediate with either a Hϩ, glyoxylate, or lipoamide bound to a putative E2, respectively
Summary
Materials—Alcohol dehydrogenase from horse liver, -mercaptoethanol, -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. Plate Reader Assays—A typical ferricyanide reductase assay reaction mixture (200 l) 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 M), 2-ketoglutarate (0 –20 mM), AcCoA (0 –2000 M), MSP (0 –2000 M), or GarA (0 –30 M). Apparent dissociation constants (Kdapp) were calculated by fitting the data to the Hill function described in Equation 8 using SigmaPlot v.10.0 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, CD3m0a2x is the maximum CD signal at saturation with MSP, [MSP] is the concentration of substrate analog, and n is the Hill coefficient. [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
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