Figure 6. Amino acid substitutions modelled in the bovine LAMAN structure.

(A) Stereographic representation of the complete structure. The active-site domain is darker than the rest of the structure. The affected residues are indicated with black balls. Mutations outside the active-site domain are labelled, and mutations in the N-terminal domain are shown in detail in (B, C). (B) Mutations affecting the folding of LAMAN. The mutated wild-type residues are shown in dark grey and the modelled mutations in white. Hydrogen bonds are displayed with broken lines and metal co-ordination with solid lines. The T355P mutation disrupts a hydrogen bond to Glu¹⁴⁹ and, probably, to the free cysteine residue Cys³⁵⁸, which in the mature enzyme participates in a disulphide bridge. Pro³⁵⁶ is in optimal position to initialize helix formation and is important for the rate of folding of LAMAN. (C) Mutations that inactivate the enzyme, but allow folding, are located close to the active site: although His²²⁰ hydrogen-bonds to the nucleophile Asp¹⁹⁶, it cannot co-ordinate the substrate as Arg²²⁰ probably does. His⁷² is involved in the metal binding, whereas Phe³²⁰ follows the active-site residue Asp³¹⁹ and makes a stacking interaction with Tyr⁸⁴. Probably, the hydrophobic stacking also stabilizes Trp⁷⁷, which is involved in substrate binding.