Structure guided engineering of a cold active esterase expands substrate range though a stabilisation mutation that allows access to a buried water chamber
Noby, N.; Johnson, R.; Tyzack, J.; Embaby, A.; Saeed, H.; Hussein, A.; Khattab, S.; Rizkallah, P.; Jones, D.
Show abstract
Cold active esterases represent an important class of enzymes capable of undertaking useful chemical transformations at low temperatures. EstN7 from Bacillus cohnii represents a true psychrophilic esterase with a temperature optimum below 20{degrees}C. We have recently determined the structure of EstN7 and have used this knowledge to understand substrate specificity and expands its substrate range through protein engineering. Substrate range is determined by a plug at the end of acyl binding pocket that blocks access to a buried water filled cavity, so limiting EstN7 to turnover of C2 and C4 substrates. Data mining revealed a potentially important commercial reaction, conversion of triacetin to only the 1,2-glyceryl diacetate isomer, which the EstN7 could achieve. Residues M187, N211 and W206 were identified as plug residues. M187 was identified as the key plug residue but mutation to alanine destabilised the structure as whole. Another plug mutation, N211A had a stabilising effect on EstN7 and suppressed the destabilising M187A mutation. The M187A-N211A variant had the broadest substrate range, capable of hydrolysing a C8 substrate. Thus, the structure of EstN7 together with focused engineering has provided new insights into the structural stability and substrate specificity that allowed expansion of substrate range.
Matching journals
The top 7 journals account for 50% of the predicted probability mass.