only the aldehyde forms and not the gem-diol forms of the specific substrate succinic semialdehyde , of selected aldehyde substrates, and of the inhibitor 3-tolualdehyde bind to the enzyme. Residue cysteine311 is crucial for their discrimination
the 3- and 4-chloro substituents as well as the 3-hydroxy substituent are all electron withdrawing. All these compounds show a reduction in reaction velocity from that of the unsubstituted benzaldehyde
NAD(P)+-dependent succinic semialdehyde dehydrogenase, BsSSADH shows similar values of the catalytic efficiency (kcat/Km) in both NAD+ and NADP+ as cofactors. The affinity of enzyme BsSSADH to NAD+ is approximately 2fold higher than that to NADP+, but the values of kcat are almost 2fold higher in the presence of NADP+ than NAD+
SpSSADH prefers NADP+ over NAD+ as a hydride acceptor, structural basis of the cofactor preference of SpSSADH, overview. Residues Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2'-phosphate group of adenine-side ribose in NADP+
Good activity with several alternate substrates including the aromatic substrates, 4-chloro- and 3-hydroxy-benzaldehyde, as well as cyclohexanal. BADH is most active with medium chain aliphatic substrates such as pentanal and hexanal.The level of activity with aliphatic substrates drops off rapidly as the chain length decreased with very little activity (<0.1%) being observed with acetaldehyde.
KM value for NAD+ increases from 0.3 to 1 mM as the pH changes from pH 5 to 10. The pH profile data are obtained at a NAD+ concentration of 5 mM, essentially saturating across the entire pH range. Similarly, data for the NADP+ pH profile are obtained at 10 mM NADP+
structures of apoenzyme and in complex with NAD+, to 1.85 and 1.9 A resolution. Two domain protein with the active site located in the interdomain interface. The NAD+ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268
purified recombinant SpSSADH in apo-form and in a binary complex with NADP+, hanging drop vapor diffusion method, for the apoform crystals: mixing 0.001 ml of protein solution with 0.001 ml of reservoir solution containing 0.1 M MES monohydrate, pH 5.8-6.1, and 18-23% w/v PEG 4000, and equilibration over 0.5 ml reservoir solution, for the binary complex crystals: mixing of protein solution with NADP+ in a 1:10 molar ratio with reservoir solution containing 0.1 M sodium acetate trihydrate, pH 4.6, and 2 M ammonium sulfate, 22Â°C, X-ray diffraction structure determination and analysis at 1.6 A and 2.1 A resolutions, respectively, molecular replacement method using the structure of Escherichia coli SSADH, PDB ID 3JZ4, as search model
Protein expression in Escherichia coli strain JM109. Single colonies are picked and the DNA is isolated and screened for the desired mutation either by restriction analysis (C103A and C249A) or by sequencing (C140A and C220A). The overall fidelity of the PCR amplification and the presence of the desired mutations are confirmed by sequencing. The plasmids containing the mutated genes are then transformed into Escherichia coli strain BL21(DE3)pLysS for protein expression.