Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

AbstractSubsite affinity maps of long substrate binding clefts in barley α-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3–12, revealed unfavorable binding energies at the internal subsites −3 and −5 and at subsites −8 and +3/+4 defining these subsites as binding barriers. Barley α-amylase 1 mutants Y105A and T212Y at subsite −6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (−2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in α-amylases

[TTK] Transglycosylations catalysed by Y151M mutant of human salivary α-amylase (HSA)

Biochemical and enzymatic characterisation has been achieved for the Tyr151Met (Y151M) mutant of human salivary α-amylase (HSA). Substantial transglycosylation capacity was detected in Y151M in addition to its hydrolytic activity. Y151M was capable of transferring maltose and maltotriose residues from a maltotetraose donor onto different 4-nitrophenyl glycosides resulting in the formation of 1-thio-β-D-glucosides, β- and α-D-glucosides and β-D-xylosides with DP 2-4 in yields up to 50%. Reactions were monitored using TLC, HPLC and MALDI-TOF MS. 1H and 13C NMR studies revealed that the Y151M preserved its stereo- and regio-selectivity. Glycosylation took place at position 4 of the glycosyl acceptors, resulting in the new α-1,4-glycosidic bonds exclusively