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

The α/β-hydrolase fold family contains mostly esterases but includes other enzymes such as hydroxynitrile lyase from Hevea brasiliensis (rubber tree, Hb HNL). Hb HNL shares 44% sequence identity and a Ser-His-Asp catalytic triad with esterase SABP2 (salicylic acid binding protein 2 from Nicotiana tabacum (tobacco)). To identify how large a region within Hb 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 Hb 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 Hb HNL and SABP2.