Biochemical and genetic analysis of a maltopentaose‐producing amylase from an alkaliphilic Gram‐positive bacterium

Abstract

Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an a-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-1 80 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical a-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial P-amylases. It appears, therefore, that A-1 80 has arisen by gene fusion events.Starch-degrading enzymes can be divided into three classes on the basis of similarities in their primary sequences. The first one comprises enzymes as different as a-amylases (EC 3.2.1.1), isoamylases (EC 3.2.1.68), pullulanases (EC 3.2.1.41) and cyclodextrin glucanotransferases (EC 2.4.1.19). aamylases are able to hydrolyze a-I ,Cglucosidic linkages within the starch molecule (endo-amylase) releasing mixtures of aanomeric glucose and malto-oligosaccharides with a certain product preference [ 1, 21. Starch-debranching enzymes like isoamylases and pullulanases, which differ only in the range of their substrates, are able to hydrolyze a-1,6-linkages at the branching points of starch [3]. Cyclodextrin glucanotransferases act as transferases which produce cyclic maltooligosaccharides by a combined endo-and exo-attack of the a-1,4-glucosidic linkages in starch [4].All of these enzymes can be placed together into one class because they share four similar regions of primary sequence [5, 61 which seem to be involved in structuring of the catalytic centre [7, 81. Furthermore, the three-dimensional structure of cyclodextrin glucanotransferases closely resembles that of aamylases [9].The second class comprises P-amylases (EC 3.2.1.2) which hydrolyze a-1 ,Clinkages from the non-reducing ends of the Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X53373. The novel amino acid sequence data have been deposited with the MIPS sequence data bank. glucose chains of starch producing maltose in the a-conformation. The deduced amino acid sequences of P-amylases exhibit high similarity in 12 well conserved regions [lo, 1 I].The third class comprises the glucoamylases (EC 3.2.1.3...

Proposed acquisition of an animal protein domain by bacteria.

“…We have now found the motif in several other published sequences of bacterial carbohydrases, including the sequences of a poly(3-hydroxybutyrate) depolymerase from Alcaligenesfaecalis (23), an exo-poly-a-D-galacturonosidase from Erwinia chrysamthemi (24), a bifunctional a-amylase-pullulanase from Clostridium thermohydrosulfuricum (25), and a maltopentaose-producing amylase from an alkaliphilic Gram-positive bacterium (26). The resemblance to Fn3 units was not described in any of these latter reports (23)(24)(25)(26).…”

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

“…The chitinase secreted by E. coli DH5␣FЈ/pCHIBT showed hydrolytic activity against the chitin tetrameric derivative (4-MU-(GlcNAc) 3 ], just a slight activity against the trimeric derivative [(4-MU-(GlcNAc) 2 ], and practically no activity against the dimeric derivative (4-MU-GlcNAc) ( Table 1) when tested at pH 6. These data indicate that ChiA74 is an endochitinase (5,22).…”

“…Therefore, the highly conserved aromatic amino acids Trp-171, Phe-177, and Trp- In relation to the second domain, this region shows homology to fibronectin type III. This protein was initially found in mammals and forms extracellular, multifunctional matrices which are important in cell bonding (28); however, it is believed that it was horizontally transferred to prokaryotes, and now it is found in bacterial chitinases, cellulases, and amylases (3,23,32). Because of the bonding action of fibronectin, its presence in these enzymes may be related to the substrate attachment.…”

Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans

“…Fi bronec tin-t ype-111-like sequences have also been found in endoglucanase B from C. jimi as three randem repeats (Meinke et al, 1991b). Homology analysis showed that domain 3 of chitinase C was similar not only to these sequences but also to amylase A-180 from an alkaliphilic eubacterium and poly(3-hydroxybutyrate) depolymerase from Alcaligenes faeqalis (Candussio et al, 1990;Saito et al, 1989). Thirteen out of 93 amino acids (14.0%) were identical in these sequences.…”