Fig 1.
Purification of cce4228 wild-type and mutants.
A. Lane M: protein marker; Lane 1–6: wild-type, Cys262Ala, Glu228Ala, Asn131Ala, Ser157Glu and Lys154Ala, respectively. B. Lane M: protein marker; Lane 1–6: Ser420Ala, Arg139Ala, Ser207Ala, Trp135Ala, Trp130Ala and Glu360Ala, respectively. 20 μg protein was deposited for both gels.
Fig 2.
The effects of chemical reagents on catalytic activity of cce4228 wild-type protein.
“+” indicates specific amounts of agents as indicated in the text were added to the assay solution, “-” indicates that agents not added to the assay solution. The catalytic initial velocity of cce4228 with 2 mM EDTA in assay solution was designated arbitrarily as 100% relative activity. The concentrations of cce4228 protein, SSA and NADP+ were fixed at 0.134 μM, 0.2 mM and 0.5 mM, respectively.
Fig 3.
Lineweaver-Burk plot of cce4228 wild-type protein with NADP+ cofactor as substrate and SSA as inhibitor.
The SSA concentrations were as labelled in the figure.
Table 1.
Kinetic parameters of cce4228 wild-type and mutants with different cofactors.
Fig 4.
The kcat and kcat/Km values of cce4228 wild-type and mutants with NADP+ as cofactor.
Fig 5.
Amino acid sequence alignment of succinic semialdehyde dehydrogenases from different sources.
SSADH sequences retrieved from the National Center for Biotechnology Information (NCBI): cce4228 protein from Cyanothece sp. ATCC51142 (ACB53576), all3556 from Anabaena sp. PCC7120 (BAB75255), a2771 from Synechococcus sp. PCC7002 (ACB00745), YneI from Samlonella typhimurium (NP_460484), YneI from E. coli (WP_115463367), GabD from E. coli (NP_417147), GabD from Human (NP_001071), GabD from A. thaliana (NP_178062), YneI from Bacillus subtilis (ARW30050). Triangle indicates residues involved in cofactor preference, and pentagram indicates residues involved in enzyme catalysis.
Fig 6.
Structural modeling of cce4228 protein.
A. The superposition of homology model of cce4228 protein and crystal structure of SSADH from Synechococcus sp. PCC7002 (PDB ID: 3VZ3), which were indicated by magenta and grey, respectively. The sticks were from the crystal structure with green (SSA) and cyan (NADP+) carbons. B. The close view of the active site. The residues with magenta and grey carbons were from homology model and crystal structure. Nitrogen, oxygen, phosphorus and sulfur atoms were colored as blue, red, orange and yellow, respectively. Residues were numbered according to the amino acid sequence of cce4228 protein. The numbers in parenthesis were from the amino acid sequence of crystal structure. The grey dashed lines indicated the possible interactions in the crystal structure. This figure was prepared by Pymol 1.503.
Fig 7.
Proposed catalytic mechanisms for SSADHs from different sources.
M1, M2 and M3 indicated the first, second and third mechanisms as stated in the text.
Fig 8.
Spectrophotometric measurements of the cysteine-NADP+ adduct in solution using cce4228 wild-type and the Cys262Ala mutant.
A. The absorbance of the cysteine-NADP+ adduct in solution using cce4228 wild-type. Spectra were recorded for 27 μM cce4228 (blue), 27 μM NADP+ (black), and a mixture of 27 μM cce4228 and NADP+ (green); B. The absorbance of the cysteine-NADP+ adduct in solution using the Cys262Ala mutant. Spectra were recorded for 27 μM Cys262Ala (blue), 27 μM NADP+ (black), and a mixture of 27 μM Cys262Ala and NADP+ (green).