Insights into the alkyl peroxide reduction pathway of Xanthomonas campestris bacterioferritin comigratory protein from the trapped intermediate-ligand complex structures.
Considerable insights into the oxidoreduction activity of the Xanthomonas campestris bacterioferritin comigratory protein (XcBCP) have been obtained from trapped intermediate/ligand complex structures determined by X-ray crystallography. Multiple sequence alignment and enzyme assay indicate that XcBCP belongs to a subfamily of atypical 2-Cys peroxiredoxins (Prxs), containing a strictly conserved peroxidatic cysteine (C(P)48) and an unconserved resolving cysteine (C(R)84). Crystals at different states, i.e. Free_SH state, Intra_SS state, and Inter_SS state, were obtained by screening the XcBCP proteins from a double C48S/C84S mutant, a wild type, and a C48A mutant, respectively. A formate or an alkyl analog with two water molecules that mimic an alkyl peroxide substrate was found close to the active site of the Free_SH or Inter_SS state, respectively. Their global structures were found to contain a novel substrate-binding pocket capable of accommodating an alkyl chain of no less than 16 carbons. In addition, in the Intra_SS or Inter_SS state, substantial local unfolding or complete unfolding of the C(R)-helix was detected, with the C(P)-helix remaining essentially unchanged. This is in contrast to the earlier observation that the C(P)-helix exhibits local unfolding during disulfide bond formation in typical 2-Cys Prxs. These rich experimental data have enabled us to propose a pathway by which XcBCP carries out its oxidoreduction activity through the alternate opening and closing of the substrate entry channel and the disulfide-bond pocket.
Liao
Shu-Ju
SJ
Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, ROC.
Yang
Chao-Yu
CY
Chin
Ko-Hsin
KH
Wang
Andrew H-J
AH
Chou
Shan-Ho
SH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry; D010545:Peroxides / Q000378:metabolism*; D054464:Peroxiredoxins / Q000378:metabolism; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism; D016959:Xanthomonas campestris / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D033903:Periplasmic Proteins; D010545:Peroxides; C068491:bacterioferritin comigratory protein, Bacteria; D013879:Thioredoxins; D009249:NADP; D010544:Peroxidases; C097049:Tpx protein, E coli; D054464:Peroxiredoxins; D003545:Cysteine