Abstract
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.
Highlights
In Escherichia coli, maltodextrins enter the periplasm preferentially via the outer membrane porin LamB [1]
Mass spectrometric molecular weight analyses using electrospray-ionization mass spectrometry (ESI-MS)1 and matrix-assisted laser-desorption/ionization mass spectrometry (MALDIMS) peptide mapping analyses were applied for identification of the molecular structure of native MalS
The function of the MalS N terminus, which is linked via a disulfide bond formed between Cys104 and Cys520 to the C-terminal amylase domain, could be to assist in folding of the amylase domain
Summary
In Escherichia coli, maltodextrins enter the periplasm preferentially via the outer membrane porin LamB [1]. Maltodextrins are either cleaved by a periplasmic amylase, MalS, or are transported to the cytoplasm via the binding protein-dependent transport complex MalEFGK2 [2]. This transport system can only transport maltodextrins up to maltoheptaose. MalS is an enzyme of 659 amino acids with a molecular mass of 74 kDa [8] It belongs to the ␣-amylase family that shares a characteristic (/␣)8-barrel domain containing the active site [9]. In this study we describe an improved purification procedure, biochemical characterization and mass spectrometric identification of the disulfide bond structure of native MalS. In our studies we used proteolytic cleavage techniques, followed by high performance liquid chromatography (HPLC) separation and MALDI-MS for the rapid identification of the disulfide bridges
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