Cyclomaltodextrinase, Neopullulanase, and Maltogenic Amylase Are Nearly Indistinguishable from Each Other

Abstract

Over 20 enzymes denoted as cyclomaltodextrinase, maltogenic amylase, or neopullulanase that share 40 -86% sequence identity with each other are found in public data bases. These enzymes are distinguished from typical ␣-amylases by containing a novel N-terminal domain and exhibiting preferential substrate specificities for cyclomaltodextrins (CDs) over starch. In this research field, a great deal of confusion exists regarding the features distinguishing the three groups of enzymes from one another. Although a different enzyme code has been assigned to each of the three different enzyme names, even a single differentiating enzymatic property has not been documented in the literature. On the other hand, an outstanding question related to this issue concerns the structural basis for the preference of these enzymes for CDs. To clarify the confusion and to address this question, we have determined the structures of two enzymes, one from alkalophilic Bacillus sp. I-5 and named cyclomaltodextrinase and the other from a Thermus species and named maltogenic amylase. The structure of the Bacillus enzyme reveals a dodecameric assembly composed of six copies of the dimer, which is the structural and functional unit of the Thermus enzyme and an enzyme named neopullulanase. The structure of the Thermus enzyme in complex with ␤-CD led to the conclusion that Trp 47 , a well conserved N-terminal domain residue, contributes greatly to the preference for ␤-CD. The common dimer formation through the novel N-terminal domain, which contributes to the preference for CDs by lining the active-site cavity, convincingly indicates that the three groups of enzymes are not different enough to preserve the different names and enzyme codes.

A remote but significant sequence homology between glycoside hydrolase clan GH‐H and family GH31

“…3) reflects the similarities and differences between clan GH-H and family GH31, and in addition contributes several novel findings to the evolutionary relationships known previously within the a-amylase family [10,[48][49][50][51]: (i) the circularly permuted GH70 family (glucosyltransferase and alternansucrase) is most closely related to the pullulanase subfamily of GH13 represented by pullulanase, isoamylase, maltooligosyl trehalose hydrolase and branching enzyme; (ii) the oligo-1,6-glucosidase subfamily (oligo-1,6-glucosidase, aglucosidase, dextran glucosidase, trehalose-6-phosphate hydrolase and isomaltulose synthase) is closest to the a-amylase and in a wider sense to the CGTase subfamily (CGTase and maltooligosaccharide-producing amylases); (iii) the neopullulanase subfamily (neopullulanase, cyclomaltodextrinase and maltogenic amylase) that may also contain amylopullulanase borders on a more diverse amylosucrase group including sucrose hydrolase, amylosucrase, trehalose synthase and 4-aglucanotransferase. The remaining four GH13 specificities, labelled as the sucrose phosphorylase group (maltooligosyl trehalose synthase, maltosyltransferase, sucrose phosphorylase and glucan debranching enzyme) are either on independent or long branches (Fig.…”

Function and Tertiary- and Quaternary-structure of Cyclodextrin-hydrolyzing Enzymes (CDase), a Group of Multisubstrate Specific Enzymes Belonging to the α-Amylase Family

“…Dimerization and dodecamerization of CDase. 5) lytic residues are located at the bottom of the groove. The shape of the active site cleft of Thermus CDase explains much slower hydrolysis of starch than CD by the enzyme.…”

“…Apparently the earlier sub classification into three enzyme classes, CDase, maltogenic amylase, and neopullulanase, is not reflected by distinct sequence differences. 5) Multiple protein sequence alignment of GH13 and related enzymes of different origin revealed the existence of four highly conserved regions in their primary sequence which contain the amino acids that form the catalytic site, as well as some amino acids that are essential for the stability of the conserved ( )8 barrel topology. 26) The alignment of CDases (Fig.…”

“…Those enzymes exhibit 34 56% sequence identity with each other and share four highly conserved regions (I, II, III and IV) in the primary amino acid sequences. 5) In addition, Podkovyrov and Zeikus 6) reported that there is an additional conserved sequence between the third and fourth conserved regions of various cyclodextrin hydrolyzing enzymes. This finding is highly relevant since the cyclodextrin binding site is known to be situated in these regions.…”

Association of Novel Domain in Active Site of Archaic Hyperthermophilic Maltogenic Amylase from Staphylothermus marinus

“…These enzyme specificities were proposed to establish the so-called neopullulanase subfamily of the ␣-amylase family GH13 (10), which is currently classified as the subfamily GH13_20 (11). CD-hydrolyzing enzymes possess a common domain at the N terminus (N domain) that is involved in substrate binding through a domain-swapped homodimeric structure (12)(13)(14)(15)(16). The narrow, deep active site generated by the N domain from another subunit in the dimer is responsible for the substrate preference, for example, for small substrates and CDs.…”