Tuning Selectivity in CalA Lipase: Beyond Tunnel Engineering

Abstract

Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications.

Structure of Dimeric Lipoprotein Lipase Reveals a Pore for Hydrolysis of Acyl Chains

“…The hydrophobic pore could provide an additional layer of substrate discrimination for mammalian lipases beyond the previously identified lid peptide and hydrophobic binding pocket. In that regard, the pore might utilize a similar specificity mechanism to the tunnel seen in C. rugosa lipase and other homologs (53, 54). This could provide an explanation for why LPL preferentially hydrolyzes the sn-1 and sn-3 position on triglycerides, as the sn-2 acyl chain interacts in a separate hydrophobic pocket facilitating enhanced substrate recognition of the sn-1 or sn-3 position by the hydrophobic pore.…”

Unusual catalytic strategy by non-heme Fe(ii)/2-oxoglutarate-dependent aspartyl hydroxylase AspH

“…The results align with earlier studies that demonstrated the role of dynamics for substrate binding, catalysis and as a valuable tool for enzyme engineering together with other computational and experimental approaches. 99,136,137,197–203…”

“…The results align with earlier studies that demonstrated the role of dynamics for substrate binding, catalysis and as a valuable tool for enzyme engineering together with other computational and experimental approaches. 99,136,137,[197][198][199][200][201][202][203] How the substrate Asp103 hFX binding mode A inuence the catalytic reaction of dioxygen activation? The high spin quintet state of Fe(III)-O-Oc − is reported to be the most favorable spin state for dioxygen activation in non-heme dioxygenases.…”

Selective Esterification Design of Lipases for TAG Synthesis Based on the Unique Structure of Curved DHA

“…This process leads to decreased fatty acid utilization in the final product. 26 However, there is a paucity of research on substrate selectivity between n-3 PUFAs and saturated fatty acids with the same chain length, especially regarding the important n-3 PUFA, DHA. The primary hindrance is the limited research on precise structural differences among substrates.…”

“…Fortunately, ongoing studies aim to enhance the lipase selectivity for long-chain fatty acids. Alejaldre et al investigated mutants carrying substitutions within the substrate tunnel and distal regions, yielding the Candida antarctica lipase A (CalA) variant LS_66 (A32T/L172M/R262H/L274W/G432D) with selectivity for long-chain palmitic acid . However, there is a paucity of research on substrate selectivity between n-3 PUFAs and saturated fatty acids with the same chain length, especially regarding the important n-3 PUFA, DHA.…”