Crystal structure of metagenomic β-xylosidase/ α-l-arabinofuranosidase activated by calcium

Abstract

The crystal structure of metagenomic β-xylosidase/α-l-arabinofuranosidase CoXyl43, activated by calcium ions, was determined in its apo and complexed forms with xylotriose or l-arabinose in the presence and absence of calcium. The presence of calcium ions dramatically increases the kcat of CoXyl43 for p-nitrophenyl β-d-xylopyranoside and reduces the Michaelis constant for p-nitrophenyl α-l-arabinofuranoside. CoXyl43 consists of a single catalytic domain comprised of a five-bladed β-propeller. In the presence of calcium, a single calcium ion was observed at the centre of this catalytic domain, behind the catalytic pocket. In the absence of calcium, the calcium ion was replaced with one sodium ion and one water molecule, and the positions of these cations were shifted by 1.3 Å. The histidine-319 side chain, which coordinates to the 2-hydroxyl oxygen atom of the bound xylose molecule in the catalytic pocket, also coordinates to the calcium ion, but not to the sodium ion. The calcium-dependent increase in activity appears to be caused by the structural change in the catalytic pocket induced by the tightly bound calcium ion and coordinating water molecules, and by the protonation state of glutamic acid-268, the catalytic acid of the enzyme. Our findings further elucidate the complex relationship between metal ions and glycosidases.

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 β-1,4-xylosidase from Geobacillus thermoleovorans IT-08

“…Fig 4C ) or arabinose (Xyl•arabinose•xylose structure; Fig 1B ) are present. This observation is in contrast with the structure of the type I GH43 β-xylosidase/α- L -arabinofuranosidase CoXyl43, in which a xylose molecule was found in subsite -1 of the enzyme [ 36 ].…”

“…This result differs from kinetic studies with S . ruminantium β-1,4-xylosidase [ 11 , 22 ] and structural studies with a metagenomic β-xylosidase/α- L -arabinofuranosidase CoXyl43 [ 36 ], a type I GH43 enzyme, which suggested that arabinose could bind to both subsites -1 and +1.…”

Insight into the cold adaptation and hemicellulose utilization of Cladosporium neopsychrotolerans from genome analysis and biochemical characterization

“…The parameters related to structure flexibility of Xyn10A, Xyn11A and Xyl43A and their structure-resolved counterparts were analyzed and shown in Table 7 . The GH10 xylanase of Thermoascus aurantiacus ( Ta Xyn10) 53 , 54 and GH11 xylanase of Thermomyces lanuginosus ( Tl Xyn11) 55 are thermophilic, while the GH43 xylosidase-arabinofuranosidase from a compost metagenome (CoXyl43) 56 , 57 is mesophilic. Their structure elements, protein interaction and accessible surface area were compared to reveal the cold-adapted strategies.…”

“…The enzyme flexibility is characterized by decreased contents of Arg and Pro reduced interactions, and increased Gly content, Gly/Pro ratio, hydrophobic accessible area 9 – 11 , 76 . To identify the cold-active catalytic mechanism of the xylanolytic enzyme system of C. neopsychrotolerans SL-16, three thermophilic or mesophilic counterparts Ta Xyn10 53 , 54 , Tl Xyn11 55 , and CoXyl43 56 , 57 were selected for comparison of some structural features (Table 7 ). Of the three xylanolytic enzymes, only Xyn10A followed the cold-adapted strategy that high Gly content, increased Gly/Pro ratio and surface hydrophobicity, and less Arg and Pro contribute to enzyme flexibility 11 .…”

“…Three structure-resolved thermophilic or mesophilic homologues, the GH10 xylanase of T. aurantiacus ( Ta Xyn10; pdb: 2BNJ) 53 , GH11 xylanase of T. lanuginosus ( Tl Xyn11; pdb: 1YNA) 55 , and GH43 xylosidase-arabinofuranosidase of a metagenome ( Co Xyl43; pdb: 5GLK) 57 , were selected for the analysis of cold-adapted strategies. Homology modeling of Xyn10A, Xyn11A and Xyl43A was conducted by using the Swiss-modeler ( https://swissmodel.expasy.org/interactive ).…”