Biochemical characterization and mass spectrometric disulfide bond mapping of periplasmic alpha-amylase MalS of Escherichia coli.
Periplasmic alpha-amylase of Escherichia coli, the malS gene product, hydrolyzes linear maltodextrins. The purified enzyme exhibited a Km of 49 microM and a Vmax of 0.36 micromol of p-nitrophenylhexaoside hydrolyzed per min per mg of protein. Amylase activity was optimal at pH 8 and was dependent on divalent cations such as Ca2+. MalS exhibited altered migration on SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Analytical ultracentrifugation and electrospray mass spectrometry indicated that MalS is monomeric. The four cysteine residues are involved in intramolecular disulfide bonds. To map disulfide bonds, MalS was proteolytically digested. The resulting peptides were separated by reverse phase-high performance liquid chromatography, and matrix-assisted laser desorption/ionization mass spectrometry analysis indicated the presence of two disulfide bonds, i.e. Cys40-58 and Cys104-520. The disulfide bond at Cys40-58 is located in an N-terminal extension of about 160 amino acids which has no homology to other amylases but to the proposed peptide binding domain of GroEL, the Hsp60 of E. coli. The N-terminal extension is linked to the C-terminal amylase domain via disulfide bond Cys104-520. Reduction of disulfide bonds by dithiothreitol treatment led to aggregation suggesting that the N terminus of MalS may represent an internal chaperone domain.
Spiess
C
C
Fakultät für Biologie, Universität Konstanz, 78457 Konstanz, Federal Republic of Germany.
Happersberger
H P
HP
Glocker
M O
MO
Spiess
E
E
Rippe
K
K
Ehrmann
M
M
D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004268:DNA-Binding Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; C058976:MalT protein, E coli; D014157:Transcription Factors; D000681:Amylases; C108136:MalS protein, E coli