Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

Abstract

Currently known fungal a-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal a-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal a-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall a-glucan. To study the biochemical characteristics of this novel cluster of a-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg "1 ), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall a-glucan synthesis is discussed. INTRODUCTIONa-Amylases are widely occurring enzymes which hydrolyse the a-(1,4)-glycosidic bonds in starch and glycogen, producing short maltooligosaccharides and maltose. Based on sequence similarity, most a-amylases (EC 3.2.1.1) are classified in glycoside hydrolase (GH) family 13, although some a-amylases originating from extremophilic organisms belong to family GH57 (Henrissat, 1991;Henrissat & Bairoch, 1996) (see also the CAZy website, http://www.cazy.org). Based on a phylogenetic analysis of 1691 different members of the GH13 family, the family has recently been divided into 35 subfamilies, all acting on aglycosidic bonds (Stam et al., 2006). Several of these subfamilies display a-amylase specificity, but many other enzyme reaction specificities are also represented. The tertiary structure of these enzymes is characterized by a (b/a) 8 barrel containing four highly conserved amino acid regions that form the active site (MacGregor et al., 2001), but the overall sequence similarity can be as low as 10 % and only a catalytic triad of amino acids is conserved invariantly (Machovic & Janecek, 2003). The shared (b/a) 8 barrel structure and catalytic mechanism within GH13 enzymes are believed to represent a common evolutionary origin (Kuriki & Imanaka, 1999;Janecek, 1997). The presence of the four conserved regions and a common secondary and tertiary structure allow construction of alignments and phylogenetic studies within the family. The phylogeny of a-amylases is generally in agreement with their origin, e.g. all fungal a-amylases are more related to each other than to the a-amylases originating from plants or animals. a-Amylases from bacteria, however, are scattered over several clusters, which group with animal, plant or fungal a-amylases, or form a separate branch (Janecek, 1994).Several a-amylases from yeasts and fungi have been studied previously (see e.g....

Identification and Phylogenetic Characterization of a New Subfamily of α-Amylase Enzymes from Marine Microorganisms

“…Most of the sequences were referred to published data (Da Lage et al 2004;Hostinová et al 2010;Stam et al 2006;van der Kaaij et al 2007). The evolutionary tree was calculated with the neighbor-joining method (Saitou and Nei 1987) implemented in the ClustalX package using the final alignment including the gaps (van der Kaaij et al 2007). The tree was displayed with the program TreeView (Page 1996).…”

“…Based on the previously reported classification of α-amylases in GH13 (Da Lage et al 2004;Hostinová et al 2010;Stam et al 2006;van der Kaaij et al 2007), we chose 54 amino acid sequences of known α-amylases to represent the 10 established subfamilies. These proteins together with AmyP and its four homologues were used for analyzing the sequence features (Fig.…”

“…Asterisks Four invariant residues, gray catalytic aspartates and glutamate, black functional histidines (Machovič and Janeček 2003;Da Lage et al 2004;MacGregor et al 2001). The alignment of α-amylases was performed for the sequence segments (approximately 300 residues) spanning the region from β1 to β8 strands of the catalytic (β/α) 8 -barrel domain (Hostinová et al 2010;van der Kaaij et al 2007). A total of 54 sequence segments were extracted and used for phylogenetic tree construction.…”

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…Genome-mining in Aspergillus niger also identified α-glucan-acting enzymes phylogenetically annotated to GH13 1, but surprisingly these contained glycosylphosphatidylinositol (GPI)-anchor sequence motifs belonging to intracellular glucanotransferases and hydrolases or they were clustered to the family GH13 5 having no previous assignments, which by cloning and recombinant enzyme production was found to contain intracellular hydrolases with low activity (van der Kaaij et al 2007a). Homologues of these intracellular enzymes are seen in genome sequences of all filamentous fungi studied.…”

Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability

“…It is clear that the α-amylases from both Archaea and plants occupy the adjacent branches only if the evolutionary tree is based on the best conserved segments or eventually larger, but conserved parts of their sequences, i.e. the so-called conserved sequence regions (Janecek et al 1999;Da Lage et al 2004;van der Kaaij et al 2007) or catalytic TIM-barrels but ignoring the positions with gaps in the alignments (Fig. 2a).…”