The effect of disulfide bond on the conformational stability and catalytic activity of beta-propeller phytase.
While beta-propeller phytases (BPPs) from Gram-positive bacteria do not carry disulfide bonding, their counterparts from Gram-negative bacteria contain cysteine residues that may form disulfide bonds. By molecular modeling, two amino acid residues of B. subtilis 168 phytase (168PhyA), Ser-161 and Leu-212, were mutated to cysteine residues. Although the double cysteine mutant was secreted from B. subtilis at an expression level that was 3.5 times higher than that of the wild type, the biochemical and enzymatic properties were unaltered. In CD spectrometric analysis, both enzymes exhibited similar apparent melting temperatures and mid-points of transition under thermal and guanidine hydrochloride induced denaturation, respectively. In enzyme assays, the mutant phytase exhibited a poor refolding ability after thermal denaturation. We postulate that the disulfide bond in BPP sequences from Gram-negative bacteria is beneficial to their stability in the periplasmic compartment. In contrast, the lack of periplasmic space in Bacillus species and the fact that Bacillus BPPs are released extracellularly may render disulfide bonds unnecessary. This may explain why in evolution, BPPs in Bacillus species do not carry disulfide bonds.
Cheng
Chiwai
C
Department of Zoology, the University of Hong Kong, Pokfulam Road, Hong Kong, China.
Wong
Kam-Bo
KB
Lim
Boon L
BL
D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D004220:Disulfides; D012694:Serine; D010832:6-Phytase; D007930:Leucine; D019791:Guanidine