Pierce_2025_bioRxiv__

The alpha/beta-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 A 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 A 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 C atoms move from their positions in Hb HNL to more closely match those in SABP2 (RMSD = 0.51 A over 235 C atoms for HNL40, 0.41 A over 219 C atoms for HNL71) and even more closely in the region within 10 A of the substrate-binding site (RMSD = 0.38 A over 58 C atoms for HNL40, 0.28 A over 53 C 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. SYNOPSIS: Variants HNL40 and HNL71 of hydroxynitrile lyase from Hevea brasiliensis 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. PDB reference: hydroxynitrile lyase from Hevea brasiliensis with forty mutations, 8SNI, hydroxynitrile lyase from Hevea brasiliensis with seventy-one mutations, 9CLR.