Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase fromPyrococcussp. ST04

Abstract

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

In silico analysis of the α-amylase family GH57: eventual subfamilies reflecting enzyme specificities

“…Of these, 1568 sequences were retrieved from the family GH57 of the CAZy database directly, whereas remaining 34 sequences of the specificity of maltogenic amylase were obtained using the BLAST. This was because the three biochemically characterized maltogenic amylases have still not been classified within the family GH57, although previous in silico analysis (Blesak and Janecek 2013 ) along with cloning, sequencing and structural studies (Comfort et al 2008 ; Jeon et al 2014 ; Jung et al 2014 ; Park et al 2014 ) have clearly suggested they exhibit all sequence/structural features characteristic of the family GH57.…”

“…Sequences were collected according to the information for the family GH57 in the CAZy database (Lombard et al 2014 ), except for the specificity of maltogenic amylase (or maltose-forming amylases) that as yet has not been assigned to any CAZy family despite the fact that it was demonstrated to exhibit all the sequence-structural features characteristic of the family GH57 (Blesak and Janecek 2013 ; Jeon et al 2014 ; Jung et al 2014 ; Park et al 2014 ). The sequences of maltogenic amylases, currently kept in CAZy among the “non-classified” sequences, were obtained by protein BLAST search (Altschul et al 1990 ) using the sequence of maltogenic amylase from Pyrococcus sp.…”

“…a Characterized GH57 enzymes: α-amylase (1)— Methanocaldococcus jannaschii (Kim et al 2001 ); 4-α-glucanotransferases (5)— Dictyoglomus thermophilum (Fukusumi et al 1988 ; Nakajima et al 2004 ), Pyrococcus furiosus (Laderman et al 1993a , b ), Thermococcus kodakaraensis (Tachibana et al 1997 ), Thermococcus litoralis (Jeon et al 1997 ; Imamura et al 2003 ), Archaeoglobus fulgidus (Labes and Schonheit 2007 ; Paul et al 2015 ); amylopullulanases (8)— Pyrococcus furiosus (Dong et al 1997 ), Thermococcus hydrothermalis (Erra-Pujada et al 1999 ), Thermococcus litoralis (Imamura et al 2004 ), Spirochaeta thermophila (Angelov et al 2010 ), Dictyoglomus turgidum (Brumm et al 2011 ), Thermococcus siculi (Jiao et al 2011 ), Thermococcus kodakaraensis (Guan et al 2013 ), Sulfolobus acidocaldarius (Choi and Cha 2015 ); amylopullulanases–cyclomaltodextrinases (4)— Staphylothermus marinus (Li et al 2013 ), Caldivirga maquilingensis (Li and Li 2015 ), Desulfurococcus amylolyticus (Park et al 2018 ), Thermophilum pendens (Li et al 2018 ); maltogenic amylases (3)— Pyrococcus furiosus (Comfort et al 2008 ), Pyrococcus sp. ST04 (Jung et al 2014 ; Park et al 2014 ), Thermococcus cleftensis (Jeon et al 2014 ); α-galactosidases (1)— Pyrococcus furiosus (van Lieshout et al 2003 ); non-specified amylases (1)—uncultured bacterium (Wang et al 2011 ); and α-glucan branching enzymes (4)— Thermococcus kodakarensis (Murakami et al 2006 ; Santos et al 2011 ), Thermotoga maritima (Ballschmiter et al 2006 ; Dickmanns et al 2006 ), Thermus thermophilus (Palomo et al 2011 ), Pyrococcus horikoshii (Na et al 2017 <...>…”

“…The family GH57 possesses its own basic characteristics that discriminate it from the family GH13 as follows: (1) the catalytic domain adopts the fold of the so-called incomplete TIM-barrel, i.e. a seven-stranded (β/α) 7 -barrel (Imamura et al 2003 ; Dickmanns et al 2006 ; Palomo et al 2011 ; Santos et al 2011 ; Park et al 2014 ; Na et al 2017 ); (2) the catalytic machinery consists of two residues—a glutamic acid as a catalytic nucleophile and an aspartic acid as a proton donor located at the strands β4 and β7, respectively, of the incomplete TIM-barrel (Imamura et al 2001 ; Palomo et al 2011 ); and (3) there are five CSRs representing the “sequence fingerprints” of the family GH57 members (Zona et al 2004 ; Blesak and Janecek 2012 ). Both families GH57 and GH13, however, are similar to each other in employing the same retaining reaction mechanism (Rye and Withers 2000 ).…”

Acceptor dependent catalytic properties of GH57 4-α-glucanotransferase from Pyrococcus sp. ST04

“…Interestingly, structural comparison with GH57 maltose-forming amylase and Branching enzyme suggested that the location of Y181 and F185 residues were similarly overlapped with F218 of GH57 maltose-forming amylase and F270 of Branching enzyme ( Supplementary Figure S5 ). F218 residue of maltose-forming amylase was known as a modulator of substrate binding, whereas F270 residue of Branching enzyme was gate-keepers on the entrance of substrate binding ( Santos et al, 2011 ; Park et al, 2014 ).…”