Biology subjects

Kazlauskas, R. J.

Publications and source records attributed to Kazlauskas, R. J..

3 recordsLinked to original sources

bioRxiv2026

Identifying a cooperative catalytic network for efficient esterase catalysis

Active-site redesign frequently yields modest improvements because residues controlling physical steps like substrate binding and product release lie outside the active site. Efficient catalysis requires a cooperative catalytic network of residues that support both the chemical and physical steps of catalysis. Using ancestral hydroxynitrile lyase HNL1, an /{beta}-hydrolase with poor esterase activity, we tested this framework directly. Matching all active-site residues to a proficient esterase improved KM five-fold but left kcat unchanged, confirming that chemical machinery alone is insufficient. Activity-weighted sequence comparison (SigniSite) across ten homologous HNLs and esterases identified 38 positions disfavoring esterase activity. Experimental refinement yielded a minimal set of fifteen substitutions (HNL1-15) with [~]60-fold higher kcat and [~]400-fold higher kcat/KM. Single-substitution reversion analysis confirmed that all fifteen substitutions are essential and provided evidence for strong cooperativity between them. X-ray crystal structures of HNL1 and HNL1-15 reveal three coordinated structural changes: reshaping the substrate-binding pocket to favor productive ester binding, restoring access to the oxyanion hole, and opening an additional tunnel for product egress and water entry. These changes arise through backbone rearrangements and altered flexibility rather than direct active-site contacts, explaining why the responsible positions escape conservation-based detection. Because cooperativity masks individual contributions, engineering such networks may require step-specific assays -- measuring binding, acylation, or product release directly -- rather than screening composite kcat.

biochemistry↗

bioRxiv2025

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

The /{beta}-hydrolase fold family contains mostly esterases but includes other enzymes such as hydroxynitrile lyase from Hevea brasiliensis (rubber tree, HbHNL). HbHNL 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 HbHNL 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[a] atoms move from their positions in HbHNL to more closely match those in SABP2 (RMSD = 0.51 [A] over 235 C[a] atoms for HNL40, 0.41 [A] over 219 C[a] 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[a] atoms for HNL40, 0.28 [A] over 53 C[a] atoms for HNL71). The pattern of tunnels in HNL40 and HNL71 are similar to each other and intermediate between the pattern in HbHNL and SABP2. SynopsisVariants 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

biochemistry↗

bioRxiv2023

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

bioengineering↗