Crystal structures of forty- and seventy-one-substitution variants of hydroxynitrile lyase from rubber tree.

UNLABELLED

The α/β-hydrolase fold family contains mostly esterases but includes other enzymes such as hydroxynitrile lyase from (rubber tree, HNL). HNL shares 44% sequence identity and a Ser-His-Asp catalytic triad with esterase SABP2 (salicylic acid binding protein 2 from (tobacco)). To identify how large a region within HNL influences the positions of the catalytic residues, we created variants where increasingly large regions surrounding the substrate-binding site had identical amino acid sequences to those in SABP2. Variant HNL40 contains 40 mutations (two inserted amino acid residues, 38 substitutions), shares 59% sequence identity with SABP2, and is identical in sequence to SABP2 within 10 Å of the substrate-binding site. Variant HNL71 contains 31 additional substitutions for a total of 71 changes (two insertions, 69 substitutions) and shares 71% sequence identity with SABP2. The sequences within 14 Å of the substrate-binding site are identical in SABP2 and HNL71. The crystal structures of HNL40 and HNL71 show that the positions of main chain Cɑ atoms move from their positions in HNL to more closely match those in SABP2 (RMSD = 0.51 Å over 235 Cɑ atoms for HNL40, 0.41 Å over 219 Cɑ atoms for HNL71) and even more closely in the region within 10 Å of the substrate-binding site (RMSD = 0.38 Å over 58 Cɑ atoms for HNL40, 0.28 Å over 53 Cɑ atoms for HNL71). The pattern of tunnels in HNL40 and HNL71 are similar to each other and intermediate between the pattern in HNL and SABP2.

SYNOPSIS

Variants HNL40 and HNL71 of hydroxynitrile lyase from contain 40 and 71 mutations, respectively, to make regions surrounding the substrate-binding site identical in sequence to esterase SABP2. X-ray structures reveal increasing similarities to SABP2 in HNL40 and HNL71. hydroxynitrile lyase from with forty mutations, 8SNI, hydroxynitrile lyase from with seventy-one mutations, 9CLR.