On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis

Abstract

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)+-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, 1H-NMR, and kinetic isotope effects. Our results confirmed SSA and NADP+ as substrates and demonstrated that a divalent metal, such as Mg2+, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pKa between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and 1H-NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP+ generates the thioester intermediate and [4A-2H, 4B-1H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.

Structural Basis for a Cofactor-dependent Oxidation Protection and Catalysis of Cyanobacterial Succinic Semialdehyde Dehydrogenase

“…Moreover, the 1-Cys SSADH from M. tuberculosis (Fig. 1C) also exhibited a pronounced lag phase when the enzyme was included as the final component in the reaction mixture (14), which suggests the presence of a cofactor-cysteine adduct. Our analysis further suggests that 2-Cys EcSSADH has two different mechanisms for redox-dependent regulation.…”

“…Given the redox-dependent regulation of HsSSADH, the oxidation-resistant catalytic features of Mycobacterium tuberculosis SSADH (14), and the ATP-dependent catalytic properties of Arabidopsis thaliana SSADH (15), the regulation of SSADH activity is much more complex than initially thought. The different regulatory mechanisms by which these enzymes are controlled remain to be resolved.…”

Structural, Kinetic and Chemical Mechanism of Isocitrate Dehydrogenase-1 from Mycobacterium tuberculosis

“…(11) proposed an α-ketoglutarate ferredoxin oxidoreductase that uses ferredoxin instead of NADP + as the oxidizing agent in the conversion of αKG to succinyl-CoA . There is also the γ-aminobutyric acid (GABA) shunt in which αKG is aminated to glutamate, decarboxylated to GABA, transaminated to succinic semialdehyde, and oxidized to the TCA intermediate succinate (12, 13) . However, metabolic labeling experiments by de Carvalho et al .…”

Functional γ-Aminobutyrate Shunt in Listeria monocytogenes: Role in Acid Tolerance and Succinate Biosynthesis

“…The SSA is then oxidized through the activity of succinic semialdehyde dehydrogenase (SSDH) (GabD) to form succinate (16). While the GABA shunt pathway has not been extensively studied in bacteria, it is thought to play a role in Glu metabolism, anaplerosis, and antioxidant defense (17,18). The use of arginine, ornithine, and agmatine as nitrogen sources by E. coli relies on the GABA shunt pathway, as these are converted first to putrescine and subsequently to GABA (19).…”