Per-Olof Syrén

Amidases Have a Hydrogen Bond that Facilitates Nitrogen Inversion, but Esterases Have Not

The fact that proteases/amidases can hydrolyze amides efficiently whereas esterases can not has been discussed during the last decades. By using molecular modeling we have found a hydrogen bond in the transition state for protease/amidase catalyzed hydrolysis of peptides and amides donated by the scissile NH‐group of the substrate. The hydrogen‐bond acceptor was found either in the enzyme (enzyme assisted) or in the substrate (substrate assisted). This new interaction with the NH‐hydrogen in the transition state (TS) was found in sixteen proteases/amidases, which represent ten different reaction mechanisms and eleven different folding families. Esterases lack this interaction and, therefore, they are slow in hydrolyzing amides. By mimicking the substrate‐assisted catalysis found in amidases we were able to shift reaction specificity of amide over ester synthesis of Candida antarctica lipase B one hundred fold. We propose that the hydrogen bond facilitates nitrogen inversion in amidases.

Esterases with an Introduced Amidase‐Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity

Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity

Mutant Lipase‐Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec‐Alcohols: A Thermodynamic Analysis of Enantioselectivity

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1‐phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild‐type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

Synthesis of heterocyclic terpenoids by promiscuous squalene-hopene cyclases.

PROMISCUOUS ENZYMES: The substrate promiscuity of squalene-hopene cyclases has been explored and applied in the enzyme-catalyzed synthesis of heterocyclic terpenoids. Features of this work include cyclization reactions without pyrophosphate activation, and stereospecific ring closure of substrates of varying chain length and terminal nucleophile. This provides a biocatalytic alternative to traditional chemical catalysts.

The solution of nitrogen inversion in amidases

An important mechanistic aspect of enzyme catalyzed amide bond hydrolysis is the specific orientation of the lone pair of the nitrogen of the scissile amide bond during catalysis. As discussed in the literature during the last decades, stereoelectronic effects cause the single lone pair in the formed tetrahedral intermediate to be situated in a non‐productive conformation in the enzyme active site and hence nitrogen inversion or rotation is necessary. By discussing recent mechanistic findings in the literature relevant for the conformation of the lone pair of the reacting amide nitrogen atom, it will be demonstrated that nature has evolved at least two catalytic strategies to cope with the stereoelectronic constraints inherent to amide bond hydrolysis regardless of the fold or catalytic mechanism. One solution to the inversion problem is to stabilize the transition state of inversion by hydrogen bond formation; another is to introduce a concerted proton shuttle mechanism that avoids inversion and delivers a hydrogen to the lone pair. By using molecular modeling it is demonstrated that the H‐bond strategy is general and can be expanded to include many amidases/proteases with important metabolic functions, including the proteasome. Some examples of the proton shuttle mechanism will also be mentioned. To complete the picture of efficient enzyme catalyzed amide bond hydrolysis, general interactions in the active site of these catalysts will be discussed. An expanded knowledge of the prerequisites of efficient amide bond hydrolysis beyond the oxyanion hole and the catalytic dyad/triad will be of importance for enzyme and drug design.

Substrate Conformations Set the Rate of Enzymatic Acrylation by Lipases

Acrylates represent a class of α,β‐unsaturated compounds of high industrial importance. We investigated the influence of substrate conformations on the experimentally determined reaction rates of the enzyme‐catalysed transacylation of methyl acrylate and derivatives by ab initio DFT B3LYP calculations and molecular dynamics simulations. The results supported a least‐motion mechanism upon the sp2 to sp3 substrate transition to reach the transition state in the enzyme active site. This was in accordance with our hypothesis that acrylates form productive transition states from their low‐energy s‐sis/s‐trans conformations. Apparent kcat values were measured for Candida antarctica lipase B (CALB), Humicola insolens cutinase and Rhizomucor miehei lipase and were compared to results from computer simulations. More potent enzymes for acryltransfer, such as the CALB mutant V190A and acrylates with higher turnover numbers, showed elevated populations of productive transition states.

Entropy is Key to the Formation of Pentacyclic Terpenoids by Enzyme-Catalyzed Polycyclization†

Polycyclizations constitute a cornerstone of chemistry and biology. Multicyclic scaffolds are generated by terpene cyclase enzymes in nature through a carbocationic polycyclization cascade of a prefolded polyisoprene backbone, for which electrostatic stabilization of transient carbocationic species is believed to drive catalysis. Computational studies and site-directed mutagenesis were used to assess the contribution of entropy to the polycyclization cascade catalyzed by the triterpene cyclase from A. acidocaldarius. Our results show that entropy contributes significantly to the rate enhancement through the release of water molecules through specific channels. A single rational point mutation that results in the disruption of one of these water channels decreased the entropic contribution to catalysis by 60 kcal mol(-1) . This work demonstrates that entropy is the key to enzyme-catalyzed polycyclizations, which are highly relevant in biology since 90 % of all natural products contain a cyclic subunit.

Squalene-hopene cyclases — evolution, dynamics and catalytic scope

Herein we highlight recent mechanistic findings on the impact of solvent dynamics on catalysis displayed by squalene-hopene cyclases (SHCs). These fascinating biocatalysts that appeared early during the evolution of terpene biosynthetic machineries exploit a catalytic aspartic acid donating the anti-oriented proton to the terminal CC double bond of pre-folded isoprenoid substrates. We review how the unusual strength of this Brønsted acid can be used to harness a plethora of non-natural protonation-driven reactions in a plastic enzyme fold. Moreover, recent results underline how the reaction termination by deprotonation or water addition is governed by the spatial location of water in the active site. Site-directed mutagenesis of amino acids located in the hydrophobic binding pocket allows for the generation of novel catalytic function by active site reshaping with relatively small enzyme libraries. A deepened understanding of triterpene cyclase dynamics in concert with chemical expertise thus have a great potential to allow for the biocatalytic manufacturing of tailored building bricks that would expand the chemical repertoire currently found in nature.

Proton Shuttle Mechanism in the Transition State of Lipase‐Catalyzed N‐Acylation of Amino Alcohols

An increased reaction rate for lipase‐catalyzed N‐acylation of amino alcohols relative to that of monofunctionalized amines can be explained by a hydrogen shuttling mechanism that avoids nitrogen inversion in the transition state. The mechanism does not involve acyl migration from an ester intermediate that would be formed first, an explanation that permeates the literature. Our suggested reaction mechanism is dependent on the preference of amino alcohols to form intramolecular hydrogen bonds and the capability of the enzyme to accommodate and exploit the specific hydrogen bonding pattern provided by the ligand during catalysis. Our proposed proton shuttle mechanism involves the transfer of two protons in the transition state concomitant with a nucleophilic attack on the acyl enzyme and provides an explanation for the high reaction rate and chemoselectivity for lipase‐catalyzed N‐acylation of amino alcohols. Moreover, the proton shuttle mechanism explains the increased reaction rate for the enzyme‐catalyzed N‐acylation of diamines and of methoxy‐2‐propylamine, for which O‐ to N‐acyl migration is impossible. A linear free‐energy relationship analysis based on the experimental results showed that all of our investigated difunctionalized amine substrates afforded a substrate‐assisted rate acceleration of the N‐acylation by the same reaction mechanism. Furthermore, the results of the analysis were consistent with partial proton transfer in the rate‐limiting transition state, which further supports our suggested proton shuttle mechanism.

Mutated variant of Candida antarctica lipase B in (S)-selective dynamic kinetic resolution of secondary alcohols.

An (S)-selective dynamic kinetic resolution of secondary alcohols, employing a mutated variant of Candida antarctica lipase B (CalB) gave products in 84-88% yield and in 90-97% ee.