Biology subjects

Kourist, R.

Publications and source records attributed to Kourist, R..

8 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

Full-Atom MPNN Based Redesign of Plant Dehydrogenase Enables Thermostability Enhancement Without Loss of Stereoselectivity

Protein stabilization is a "Holy Grail" of biocatalysis, and stability design is an area of intense research interest. While it is increasingly feasible to effectively increase enzyme thermostability, optimization without compromising activity or selectivity remains a significant challenge. Here, we use full-atom protein sequence design with sidechain conditioning (FAMPNN) to engineer thermostable variants of the borneol dehydrogenase from Salvia rosmarinus (SrBDH1), an enzyme from a family where unselective enzymes dominate, and selectivity is determined by dynamical considerations. By combining FAMPNN design with residue conservation analysis and avoiding active site residues, we were able to computationally design SrBDH1 variants with up to 10 {degrees}C enhanced thermostability and strongly increased half-life time at elevated temperature, while retaining selectivity towards (+)-borneol. This design framework, integrating de novo and physics-based protein design tools, demonstrates that stability can be enhanced without disrupting functionally relevant dynamics, providing a route to engineer robust and selective biocatalysts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/719482v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@1a35073org.highwire.dtl.DTLVardef@f6c56dorg.highwire.dtl.DTLVardef@11b965forg.highwire.dtl.DTLVardef@2d6eef_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

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↗

bioRxiv2025

Deciphering the evolutionary origin of the stereoselectivity of short-chain dehydrogenases in the oxidation of the monoterpenol 1-borneol

Enzyme engineering has produced numerous methods to optimize enzymes for biotechnological processes; however, less is known about how natural evolution creates new functionalities. We investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases (BDHs), which feature hydrophobic active-sites and are enantioselective towards dibornane-type monoterpenols. Ancestral sequence reconstruction provided a trajectory from the oldest unselective BDH ancestor N30 (E=12) toward a more recent selective ancestor N32, involving 19 mutations: 18 mutations are peripheral, one (I111L) occurs in the active-site. The mutation L111I in the hydrophobic pocket increased the selectivity of N30, while the back-mutation I111L decreased the selectivity of N32. Additional peripheral mutations (V136L/G169A/V183I) were required for high selectivity. Crystal structures suggested that protein dynamics, rather than structural changes shape these catalytic properties; this was confirmed by ML/MM simulations of ligand binding. Funnel-metadynamics simulations revealed a correlation between the active-sites solvent-accessible surface area (SASA) and selectivity. This potential evolutionary pathway shapes enantioselectivity, and guides future enzyme engineering campaigns.

biochemistry↗

bioRxiv2024

Engineering of Transmembrane Alkane Monooxygenases to Improve a Key Reaction Step in the Synthesis of Polymer Precursor Tulipalin A

The -methylene-{gamma}-butyrolactone tulipalin A, naturally found in tulips can polymerize via addition at the vinyl group or via ring-opening polymerization, making it a highly promising monomer for biobased polymers. As tulipalin A biosynthesis in plants remains elusive, we propose a pathway for its synthesis starting from the metabolic intermediate isoprenol. For this, terminal hydroxylation of the -methylene substrate isoprenyl acetate is a decisive step. While a panel of fungal unspecific peroxygenases showed a preference for the undesired epoxidation of the exo-olefin group, bacterial alkane monooxygenases were specific for terminal hydroxylation. A combination of protein engineering based on de novo structure prediction of the membrane enzymes with cell engineering allowed to increase the specific activity by 6-fold to 1.83 U gcdw -1, unlocking this reaction for the fermentative production of tulipalin A from renewable resources.

bioengineering↗

bioRxiv2024

Strong heterologous electron sink outcompetes alternative electron transport pathways in photosynthesis

Improvement of photosynthesis requires a thorough understanding of electron partitioning under both natural and strong electron sink conditions. We applied a wide array of state-of-the-art biophysical and biochemical techniques to thoroughly investigate the fate of photosynthetic electrons in the engineered cyanobacterium Synechocystis sp. PCC 6803, a blueprint for photosynthetic biotechnology, expressing the heterologous gene for ene-reductase, YqjM. This recombinant enzyme catalyses the reduction of an exogenously added substrate into the desired product by utilising photosynthetically produced NAD(P)H, enabling whole-cell biotransformation. Through coupling the biotransformation reaction with biophysical measurements, we demonstrated that the strong artificial electron sink, outcompetes the natural electron valves, the flavodiiron protein-driven Mehler-like reaction, and cyclic electron transport. These results show that ferredoxin-NAD(P)H-oxidoreductase (FNR) is the preferred route for delivering photosynthetic electrons from reduced ferredoxin and the cellular NADPH/NADP+ ratio as a key factor in orchestrating photosynthetic electron flux. These insights are crucial for understanding molecular mechanisms of photosynthetic electron transport and harnessing photosynthesis for sustainable bioproduction by engineering the cellular source/sink balance. Furthermore, we conclude that identifying the bioenergetic bottleneck of a heterologous electron sink is a crucial prerequisite for targeted engineering of photosynthetic biotransformation platforms. Significance statementWe coupled the photosynthetic and biocatalytic (whole-cell biotransformation) performance of model cyanobacteria. We employed a heterologous NAD(P)H utilising enzyme, as a strong artificial electron sink, allowing us to gain a comprehensive understanding of photosynthetic electron partitioning. We demonstrated that the strong electron sink outcompetes natural electron sinks and cyclic electron transport.

plant biology↗

bioRxiv2021

Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 [A] resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

biophysics↗