Jürgen Pleiss

Anatomy of lipase binding sites: the scissile fatty acid binding site.

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.

The Lipase Engineering Database: a navigation and analysis tool for protein families

The Lipase Engineering Database (LED) (http://www.led.uni-stuttgart.de) integrates information on sequence, structure, and function of lipases, esterases, and related proteins. Sequence data on 806 protein entries are assigned to 38 homologous families, which are grouped into 16 superfamilies with no global sequence similarity between each other. For each family, multisequence alignments are provided with functionally relevant residues annotated. Pre-calculated phylogenetic trees allow navigation inside superfamilies. Experimental structures of 45 proteins are superposed and consistently annotated. The LED has been applied to systematically analyze sequence-structure-function relationships of this vast and diverse enzyme class. It is a useful tool to identify functionally relevant residues apart from the active site residues, and to design mutants with desired substrate specificity.

Lipase engineering database: Understanding and exploiting sequence–structure–function relationships

Abstract The Lipase Engineering Database is a WWW-accessible resource on sequence–structure–function relationships of microbial lipases. Sequences of 92 microbial lipases and homologous serine hydrolases were assigned to 32 homologous families and 15 superfamilies. Multisequence alignments of all homologous families and superfamilies are provided. Functionally relevant amino acids are annotated. The catalytic serine is part of the conserved nucleophilic elbow and was identified in all sequences by its conserved signature GxSxG. The complete catalytic machinery (catalytic triad and two oxyanion hole residues) could be annotated in 91% of LED sequence entries. Published mutants and their properties are provided. The X-ray structures of 22 lipases were superposed and consistently annotated. Sequence and structure data were applied to study the role of the first oxyanion hole residues. Although the backbone amides contribute to the oxyanion hole rather than side chains, the residues are well conserved. Two sequence signatures including the first oxyanion hole residue were identified: GX and GGGX. In the GX type, the position of the first oxyanion hole residue X is stabilized by one or several anchor residues. If X is hydrophilic, it is hydrogen bonded to hydrophilic anchor residues, while hydrophobic oxyanion hole residues bind to hydrophobic pockets. The GGGX type includes short chain length specific lipases and carboxylesterases. The first oxyanion hole residue G is stabilized by interaction of the dipeptide GX with the side chain of the second oxyanion hole residues, which is a conserved alanine as C-terminal neighbour of the catalytic serine. Thus, short chain specific lipases and carboxylesterase can be identified by combining the signatures GGGX and GxSAG. Consistently annotated aligned sequences and superimposed structures of microbial lipases help to understand the functional role of individual amino acids and thus the LED is a useful tool for protein engineering. The Lipase Engineering Database is available at http://www.led.uni-stuttgart.de/ .

Activity of Lipases and Esterases towards Tertiary Alcohols: Insights into Structure–Function Relationships†

Hydrolytic enzymes are versatile biocatalysts and find increasing applications in organic synthesis and a considerable number of industrial processes using these enzymes have been commercialized. Within this class, lipases (E.C. 3.1.1.3) and carboxyl esterases (E.C. 3.1.1.1) are frequently used as they accept a broad range of non-natural substrates, are usually very stable in organic solvents and exhibit good to excellent stereoselectivity.

Selective Catalytic Oxidation of CH Bonds with Molecular Oxygen

Although catalytic reductions, cross‐couplings, metathesis, and oxidation of CC double bonds are well established, the corresponding catalytic hydroxylations of CH bonds in alkanes, arenes, or benzylic (allylic) positions, particularly with O2, the cheapest, “greenest”, and most abundant oxidant, are severely lacking. Certainly, some promising examples in homogenous and heterogenous catalysis exist, as well as enzymes that can perform catalytic aerobic oxidations on various substrates, but these have never achieved an industrial‐scale, owing to a low space‐time‐yield and poor stability. This review illustrates recent advances in aerobic oxidation catalysis by discussing selected examples, and aims to stimulate further exciting work in this area. Theoretical work on catalyst precursors, resting states, and elementary steps, as well as model reactions complemented by spectroscopic studies provide detailed insight into the molecular mechanisms of oxidation catalyses and pave the way for preparative applications. However, O2 also poses a safety hazard, especially when used for large scale reactions, therefore sophisticated methodologies have been developed to minimize these risks and to allow convenient transfer onto industrial scale.

Modeling structure and flexibility of Candida antarctica lipase B in organic solvents

BackgroundThe structure and flexibility of Candida antarctica lipase B in water and five different organic solvent models was investigated using multiple molecular dynamics simulations to describe the effect of solvents on structure and dynamics. Interactions of the solvents with the protein and the distribution of water molecules at the protein surface were examined.ResultsThe simulated structure was independent of the solvent, and had a low deviation from the crystal structure. However, the hydrophilic surface of CALB in non-polar solvents decreased by 10% in comparison to water, while the hydrophobic surface is slightly increased by 1%. There is a large influence on the flexibility depending on the dielectric constant of the solvent, with a high flexibility in water and a low flexibility in organic solvents. With decreasing dielectric constant, the number of surface bound water molecules significantly increased and a spanning water network with an increasing size was formed.ConclusionThe reduced flexibility of Candida antarctica lipase B in organic solvents is caused by a spanning water network resulting from less mobile and slowly exchanging water molecules at the protein-surface. The reduced flexibility of Candida antarctica lipase B in organic solvent is not only caused by the interactions between solvent-protein, but mainly by the formation of a spanning water network.

Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity.

Monooxygenase mutants: A minimal and highly enriched CYP102A1 mutant library was constructed by combining five hydrophobic amino acids in two positions. The library was screened with four different terpene substrates. Eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

Carboxylesterases containing the sequence motif GGGX catalyze the hydrolysis of esters of chiral tertiary alcohols, albeit with only low to moderate enantioselectivity, for three model substrates (linalyl acetate, methyl‐1‐pentin‐1‐yl acetate, 2phenyl‐3‐butin‐2‐yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity for this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p‐nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate–enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p‐nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this amino acid for an alanine residue led to a sixfold increase of enantioselectivity (E=19) towards 2phenyl‐3‐butin‐2‐yl acetate. However, the effect of this mutation is specific: the same mutant had the opposite enantiopreference towards the substrate linalyl acetate. Thus, depending on the substrate structure, the same mutant has either increased enantioselectivity or opposite enantiopreference compared to the wild‐type enzyme.

Substrate specificity of lipase B from Candida antarctica in the synthesis of arylaliphatic glycolipids

Arylaliphatic glycolipids are known for their pharmaceutical and medicinal properties. We found that a great variety of arylaliphatic esters can be synthesized from non-activated substrates like glucose or the natural occurring drug salicin using lipase B from Candida antarctica (CAL-B). However, esters based on aromatic carboxylic acids or unsaturated arylaliphatic acids, like cinnamic acid and its derivatives, which are known to display anticancer activity, could not be obtained. In this work, we performed computer-aided molecular modeling based on data of our work published recently and syntheses of new glycolipids to understand why some substances are accepted by CAL-B while some are not. For this purpose, we investigated the accessibility of the lipase binding site for the arylaliphatic acyl donors as well as the steric interactions between the aglycons of glucosides and the residues of the alcohol binding pocket in order to elucidate potentials and limitations of CAL-B for the synthesis of aromatic glycolipids.