The loop structure of Actinomycete glycoside hydrolase family 5 mannanases governs substrate recognition

Abstract

Endo-β-1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III) ,6(IV) -α-d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 Å, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around -1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the -1 subsite.

Molecular insights into the mechanism of thermal stability of actinomycete mannanase

“…A, open circle) . The amino acid sequences in loop7 differed between StMan and these mannanases; however, the sequences were well conserved in SlMan, StManII and TfMan . There mannanases did not form a hydrogen bond between loop6 and loop7 because the residue at the position of Ser247 in loop6 of StMan was Gly in these mannanases (Fig.…”

“…Although mutation analysis showed that the loss of the hydrogen bond did not affect the binding of Ca 2+ to the mutants, it led to a decrease in the eTS. Loop7 of StMandC was positioned for a tight interaction with the substrate compared to its location in the crystal structure without substrate . Asp279, Gln280 and Trp281 in loop7 showed high B‐factors, which exhibited high flexibility and low thermal stability .…”

“…In loop7, the main chains of Gly276 and Pro284 and side chains of Asp283 and Glu286 were the Ca 2+ ‐binding residues located within 2.21–2.40 Å of the Ca 2+ (Fig. A,B), which was present outside the catalytic cleft . In addition, the crystal structures also revealed that a hydrogen bond of 2.71 or 3.34 Å was formed between Ser247 in loop6 and Asp279 in loop7, which were located inside the catalytic cleft (Fig.…”

“…These results indicate that loop7 was definitely the Ca 2+ ‐binding site. GH5_8 actinobacterial mannanases were mainly grouped into two types of loop7 conformations: StMan‐type formed a flexible loop7 by insertion of Gly276 in StMan; the other mannanases formed a short loop7 . Although loop7 conformations differed between them, both loop7s had Ca 2+ ‐binding ability .…”

Metatranscriptomic analysis of functional response of colonic microbiota to different dietary fibers in growing pigs

“…The abundance of GH5 increased along with the increase of Streptococcus and Prevotella, GH5 was mainly annotated as endo-glucanase, which is the main ingredient of cellulase [40]. and it also involved in the hydrolysis of galactose, some loops in GH5 enzymes could recognize galactosyl unit [41]. The abundance of GH16 increased along with the increase of Streptococcus and Mesorhizobium, and the degradation of galactose was predicted could be initiated by GH16 [42], this galactanases had substrate speci city acting on galactooligosaccharides [43].…”