Extended Data Fig. 9: Mechanism of ALG6-catalysed glucosyl transfer.
From: Structure and mechanism of the ER-based glucosyltransferase ALG6
a, Chemical structures of synthetic donor substrate analogues (left) and their functional analysis (right). Compound numbers are indicated in bold and in parentheses. Analysis of ALG6 activity as described in Fig. 1b but in the presence of different substrate analogues as indicated above the lanes. The lane labelled Man9-LLO is a control sample for size comparison. In one of the ALG6 reactions, a pre-incubation of ALG6 with 10 mM EDTA was used to remove any divalent ions from the solution. b, Proposed three-state mechanism of ALG6; intermediate states are numbered and encircled. State 1 represents the apo state of ALG6 based on our EM structure, with ALG6 in surface representation (brown) and clipped for better visualization of the substrate-binding pocket. State 2 represents ALG6 bound to a donor substrate based on our EM structure, with Dol25-P-Glc shown in sphere representation with carbon atoms coloured green and oxygen atoms coloured red. State 3 represents a putative ternary complex based on the apo structure of ALG6, with substrates drawn manually. The inset depicts the proposed reaction mechanism, with Asp69 acting as a general base that deprotonates the C3 hydroxyl group of the attacking mannose moiety.
