Biology subjects

van der Pol, E.

Publications and source records attributed to van der Pol, E..

4 recordsLinked to original sources

bioRxiv2026

Hydrophobic pocket engineering of arylmalonate decarboxylase expands its substrate scope towards the synthesis of the (R)-enantiomers of sterically hindered carboxylic acids

Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of -aryl and -alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity.

biochemistry↗

bioRxiv2026

Ancestors of Arylmalonate Decarboxylase show increased Activity, Stability and Stereoselectivity

Bacterial aryl malonate decarboxylase (AMDase) generates a wide spectrum of alpha-chiral carboxylic acids in outstanding optical purity, including several non-steroidal anti-inflammatory drugs and chiral building blocks. However, extant AMDase from Bordetella bronchiseptica (BbAMDase) and related enzymes of the same family have three main limitations: (i) low operational and thermal stability, (ii) limited substrate spectrum regarding the size of the smaller substituent on the alpha-C-atom, and (iii) low stereoselectivity towards alpha-alkenyl-alpha-alkyl malonic acids. To address these limitations, we expanded the structural diversity of the AMDase family by ancestral sequence reconstruction (ASR). The phylogenetic analysis of the decarboxylase revealed conserved structural motifs and key amino acids in the hydrophobic active-site cavity, a catalytic motif crucial for activity and selectivity of the enzyme. The analysis highlighted the natural distribution of amino acid exchanges previously identified via enzyme engineering. AMDase ancestors showed higher stability, activity, and, in one case, also stereoselectivity than BbAMDase. While a 10 degree higher unfolding temperature of AMDase ancestors is a frequent ASR result, ancestor N131 exhibited an exceptional 294-fold improvement in half-life time and a 1.8-fold increased specific activity (25.1 U / mg) toward 2-methyl-2-phenyl malonate in a preparative scale application. Computational analysis attributes this improved stability to a substantial increase in surface Glu side chains, which form a protective hydration shell that enhances solubility and mitigates aggregation. Ancestor N31 formed 2-methyl-but-3-enoic acid from its corresponding malonic acid in excellent optical purity (99.7% eeR). Finally, the stereoselectivity of the ancestors was completely inverted by switching the catalytic cysteine residues (G74C/C188G).

biochemistry↗

bioRxiv2025

Engineering Membrane-Bound Alkane Monooxygenase from Marinobacter sp. for Increased Activity in the Selective ω-Hydroxylation of Linear and Branched Aliphatic Esters

The regio- and stereoselective hydroxylation of unactivated C(sp3)-H bonds is an important reaction in organic synthesis. While bacterial alkane monooxygenase AlkB catalyzes the terminal hydroxylation of aliphatic esters with excellent regioselectivity, the molecular principles of substrate recognition and selectivity of this integral membrane enzyme are still poorly understood. In this study, we investigated the substrate scope and engineered the medium-chain alkane monooxygenase from Marinobacter sp. (M_AlkB) for the terminal hydroxylation of linear and branched esters of fatty acids and alcohols. For the first time, we demonstrated the stereoselectivity of AlkB towards prochiral substrates containing terminal gem-dimethyl groups, leading to the corresponding chiral {beta}-methyl primary alcohols in good optical purity (51-79% ee). The hydroxylation products can be further derivatized to chiral diols and lactones. Substitution of the highly conserved active site residue F169 to leucine increased the activity towards short and medium-chain esters up to two-fold. While the wildtype enzyme does not accept long-chain substrates, activity towards n-dodecyl acetate could be unlocked by reducing the size of the tryptophan residue 60 situated in the putative substrate tunnel. Substitution of the peripheral I238 with valine increased activity regardless of the chain length of the substrate. Our results lay the groundwork for the establishment of a whole-cell process for the regio- and stereoselective hydroxylation of linear and branched esters, leading to valuable bifunctionalized products. The insights gained from mutating key residues and the substrate acceptance of AlkB will guide future protein engineering campaigns. SIGNIFICANCEWe report rational engineering of a membrane-bound alkane monooxygenase in the hydroxylation of aliphatic esters, giving rise to diols, hydroxy acids, and lactones. By site-directed mutagenesis, we increased the activity towards short and medium-chain substrates, and we unlocked the activity towards long-chain substrates. We uncovered the enzymes capacity to discriminate between prochiral methyl groups, establishing new routes for the synthesis of chiral diols and lactones, important building blocks for the pharmaceutical industry.

biochemistry↗

bioRxiv2019

Engineered Retroviruses as Fluorescent Biological Reference Particles for Nanoscale Flow Cytometry

There has been renewed interest in the use of flow cytometry for single particle phenotypic analysis of particles in the nanometer size-range such as viruses, organelles, bacteria and extracellular vesicles (EVs). However, many of these particles are smaller than 200 nm in diameter, which places them at the limit of detection for many commercial flow cytometers. The use of reference particles of diameter, fluorescence, and light-scattering properties akin to those of the small biological particles being studied is therefore imperative for accurate and reproducible data acquisition and reporting across different instruments and analytical technologies. We show here that an engineered murine leukemia virus (MLV) can act as a fluorescence reference particle for other small particles such as retroviruses and EVs. More specifically, we show that engineered MLV is a highly monodisperse enveloped particle that can act as a surrogate to demonstrate the various effects of antibody labeling on the physical properties of small biological particles in a similar diameter range.

cell biology↗