Escherichia coli produces a cytoplasmic alpha-amylase, AmyA

Abstract

In the gap between two closely linked flagellar gene clusters on the Escherichia coi and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coi map), we found an open reading frame whose sequence suggested that it encoded an a-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other ct-amylases were to the excreted liquefying a-amylases of bacilli, with >40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coil gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the ac-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an a-amylase rather than a 1-amylase. The newly discovered gene has been named amyA.The natural physiological role of the AmyA protein is not yet evident.During an investigation of the flagellar genes ofEscherichia coli and Salmonella typhimuium, we encountered a nearby open reading frame whose deduced product sequence suggested that it was that of a cytoplasmic a-amylase.The only major cytoplasmic polysaccharide in these enteric bacteria is glycogen, which is laid down as an energy and carbon reserve, especially under conditions in which carbon is abundant but another major essential element, such as nitrogen, is limiting (27). Under normal growth conditions, glycogen represents only about 2.5% of the dry weight of the cell (26), but it can reach as high as 30% under conditions of carbon abundance and deprivation of another essential element, such as nitrogen (33). A possible role for a cytoplasmic a-amylase might therefore be in glycogen metabolism.The enzymes responsible for glycogen synthesis in E. coli have been studied extensively; they are glucose-i-phosphate adenyltransferase, glycogen synthase, and 1,4-a-glucan branching enzyme. The corresponding structural genes (glgC, glgA, and glgB) are clustered at 76 min on the map (1,2,18).Less is known about the genetics and enzymology of glycogen breakdown in E. coli. By analogy with mammalian systems, one might expect at least an a-glucan phosphorylase and a debranching enzyme. An E. coli protein with a-glucan phosphorylase activity was reported by Chen a...

Cloning, Sequencing, Characterization, and Expression of an Extracellular α-Amylase from the Hyperthermophilic ArchaeonPyrococcus furiosus in Escherichia coli andBacillus subtilis

“…8B. It reveals that the P. furiosus ␣-amylase has about 47% homology to Bacillus licheniformis (Amy-Bacli) (24), 44% to Salmonella typhimurium (Amy2-Salty) (25), 39% to Oryza sativa (Amy1-Oryza) (26), and 34% to the Pseudomonas stutzeri (Amt4-Psest) (27) enzymes.…”

“…On the basis of the DNA sequence and N terminus amino acid sequence determination of the mature ␣-amylase, the amino acid sequences of the signal peptide and of the mature ␣-amylase have been deduced. The signal peptide is 25 amino acids long and is cleaved between Ala 25 and Ala 26 . The DNA fragment containing the ␣-amylase gene encompasses 1740 nucleotides, with the initiation codon GTG at position ϩ1 (Fig.…”

“…The promoter region containing a consensus BoxA sequence TTTATA (Ϫ53 to Ϫ58) is underlined. The site of the cleavage of signal peptide is between Ala 25 and Ala 26 and indicated by a dot. Immediately following the TGA stop codon there is a pyrimidine-rich sequence (which is between 1383 and 1401 and underlined) as found in other Pyrococcus sp.…”

Sequence Similarities and Evolutionary Relationships of Microbial, Plant and Animal α‐amylases

“…2A reflects several facts already reported, such as the high degree of sequential similarity between the a-amylases of Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus stearothermophilus [ 131 (liquefying aamylases), between the A. hydrophila and Xanthomonas campestris [IS] enzymes (next to the cluster of streptomycetes and animal a-amylases), and between those from Escherichia coli and Salmonella typhimurium [25] (cytoplasmic a-amylases). Although the three above-mentioned groups of a-amylases (fungi and yeasts, plants, and streptomycetes and animals) are already described as homologous [ 14, 16 -181, their individual evolutionary trees (Fig.…”

Characterization of a genetic locus essential for maltose-maltotriose utilization in Staphylococcus xylosus

“…Uptake of maltose and maltodextrins up to maltoheptaose into the cytoplasm is mediated by a multicomponent, binding protein-dependent transport system encoded by malE, malF, malG, and malK (38). Degradation of maltodextrins is achieved by the action of three cytoplasmic enzymes, amylomaltase (21), maltodextrin phosphorylase (34), and maltodextrin glucosidase (29,43), the gene products of malQ, malP, and malZ, respectively, and a periplasmic ␣-amylase (24,36) specified by malS, which is required only for growth on long maltodextrins. None of these enzymes is able to cleave maltose itself, but they recognize maltotriose or longer maltodextrins as substrates.…”