The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A ␣-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 ␣-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. Starch is an excellent carbon and energy source for many microorganisms, which employ a dedicated set of proteins for extracellular hydrolysis of this polysaccharide, uptake of shorter oligosaccharides into the cell, and further degradation into glucose. Most studies on degradation of starch by microbial enzymes have focused on soluble starch. This has resulted in the identification and characterization of a large variety of enzymes cleaving either ␣(1¡4) or ␣(1¡6) linkages in amylose and amylopectin. Most of these enzymes belong to the glycoside hydrolase 13 (GH13) family (1). Sequence diversity is such that, at the moment, the GH13 family contains a total of 40 subfamilies (1). Most of the new members in subfamilies are identified in DNA sequencing projects, and biochemical information about the activity and specificity of these potentially new enzymes is highly lagging.Many plants produce starch in a granular form for the storage of carbohydrates. The crystallinity of such granules varies with the plant source. Potato starch granules have a relatively high degree of crystallinity, making them notoriously resistant to bacterial and fungal degradation (2-4). Nevertheless, some microorganisms have been reported to employ enzymes that are able to digest granular starch (5, 6).Amylases found to be involved in granular starch degradation are often multidomain enzymes that include one or more carbohydrate binding modules (CBMs), which aid in the binding of the enzyme to the granular substrate (7-10).In previous work, various bacteria able to grow on potato starch granules as a carbon source were isolated, and their enzymatic degradation mechanism was evaluated. Initially, this resulted in the identification of an enzyme mechanism involving peeling off layer afte...
Microbacterium aurum B8.A is a bacterium that originates from a potato starch‐processing plant and employs a GH13 α‐amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi‐modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α‐glucan substrates. Therefore, we propose that this novel starch‐binding domain is a new carbohydrate‐binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence‐based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi‐domain α‐amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α‐amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α‐amylases originating from human gut‐associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.
Fibronectin type III (FNIII) domains were first identified in the eukaryotic plasma protein fibronectin, where they act as structural spacers or enable protein-protein interactions. Recently we characterized two large and multi-domain amylases in Microbacterium aurum B8.A that both carry multiple FNIII and carbohydrate binding modules (CBMs). The role of (multiple) FNIII domains in such carbohydrate acting enzymes is currently unclear. Four hypothetical functions are considered here: a substrate surface disruption domain, a carbohydrate binding module, as a stable linker, or enabling protein-protein interactions. We performed a phylogenetic analysis of all FNIII domains identified in proteins listed in the CAZy database. These data clearly show that the FNIII domains in eukaryotic and archaeal CAZy proteins are of bacterial origin and also provides examples of interkingdom gene transfer from Bacteria to Archaea and Eucarya. FNIII domains occur in a wide variety of CAZy enzymes acting on many different substrates, suggesting that they have a non-specific role in these proteins. While CBM domains are mostly found at protein termini, FNIII domains are commonly located between other protein domains. FNIII domains in carbohydrate acting enzymes thus may function mainly as stable linkers to allow optimal positioning and/or flexibility of the catalytic domain and other domains, such as CBM.
The bacterium Microbacterium aurum strain B8.A degrades granular starches, using the multi-domain MaAmyA α-amylase to initiate granule degradation through pore formation. This paper reports the characterization of the M. aurum B8.A MaAmyB enzyme, a second starch-acting enzyme with multiple FNIII and CBM25 domains. MaAmyB was characterized as an α-glucan 1,4-α-maltohexaosidase with the ability to subsequently hydrolyze maltohexaose to maltose through the release of glucose. MaAmyB also displays exo-activity with a double blocked PNPG7 substrate, releasing PNP. In M. aurum B8.A, MaAmyB may contribute to degradation of starch granules by rapidly hydrolyzing the helical and linear starch chains that become exposed after pore formation by MaAmyA. Bioinformatics analysis showed that MaAmyB represents a novel GH13 subfamily, designated GH13_42, currently with 165 members, all in Gram-positive soil dwelling bacteria, mostly Streptomyces. All members have an unusually large catalytic domain (AB-regions), due to three insertions compared to established α-amylases, and an aberrant C-region, which has only 30% identity to established GH13 C-regions. Most GH13_42 members have three N-terminal domains (2 CBM25 and 1 FNIII). This is unusual as starch binding domains are commonly found at the C-termini of α-amylases. The evolution of the multi-domain M. aurum B8.A MaAmyA and MaAmyB enzymes is discussed.
Glucansucrase (GS) (often labelled glycosyltransferase; GTF) enzymes (EC 2.4.1.5) of lactic acid bacteria use sucrose to synthesize a diversity of a-d-glucans with a-(1fi6) (dextran, mainly found in Leuconostoc), a-(1fi3) (mutan, mainly found in Streptococcus), alternating a-(1fi3) and a-(1fi6) (alternan, only reported in Leuconostoc mesenteroides), a-(1fi4) [reuteran, by reuteransucrase from Lactobacillus reuteri 121 (GTFA) and reuteransucrase from L. reuteri ATCC 55730 (GTFO)] glucosidic bonds [1][2][3][4][5]. GTFA and GTFO show 68% sequence identity, and synthesize reuterans with approximately 50% and 70% a-(1fi4) glucosidic linkages, respectively, plus a-(1fi6) linkages ( 50% and 30%, respectively). Both enzymes also differ strongly in their transglucosylation ⁄ hydrolysis activity ratios. GTFA and GTFO hydrolyze approximately 20% and 50% of the sucrose provided, respectively [5,6].Based on the deduced amino acid sequences, GS enzymes are composed of four distinct structural domains, which, from the N-to C-terminus (Fig. 1A) The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into a-d-glucans (labelled reuterans) with mainly a-(1fi4) glucosidic linkages (50% and 70%, respectively), plus a-(1fi6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA ⁄ O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1-3; O1-3), and used to construct six additional GTFA ⁄ O hybrids. All hybrid enzymes were able to synthesize a-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2 ⁄ O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1 ⁄ O1 regions make a strong contribution. Some GTFO derived hybrid ⁄ mutant enzymes displayed strongly increased transglucosylation ⁄ hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligoand polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation ⁄ hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in a-(1fi4) and a-(1fi6) linkage ratios.
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