Detailed Structure–Function Correlations of Bacillus subtilis Acetolactate Synthase

Isobutanol is deemed to be a next‐generation biofuel and a renewable platform chemical. 1 Non‐natural biosynthetic pathways for isobutanol production have been implemented in cell‐based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst. 2 – 6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg²⁺ as cofactors. AlsS also catalyzes the conversion of 2‐ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP‐dependent enzymes. To unravel catalytically relevant structure‐function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg²⁺ and in a transition state with a 2‐lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.     

Altering the Regioselectivity of a Nitroreductase in the Synthesis of Arylhydroxylamines by Structure‐Based Engineering

Nitroreductases have great potential for the highly efficient reduction of aryl nitro compounds to arylhydroxylamines. However, regioselective reduction of the desired nitro group in polynitroarenes is still a challenge. Here, we describe the structure‐based engineering of Escherichia coli nitroreductase NfsB to alter its regioselectivity, in order to achieve reduction of a target nitro group. When 2,4‐dinitrotoluene was used as the substrate, the wild‐type enzyme regioselectively reduced the 4‐NO₂ group, but the T41L/N71S/F124W mutant primarily reduced the 2‐NO₂ group, without loss of activity. The crystal structure of T41L/N71S/F124W and docking experiments indicated that the regioselectivity change (from 4‐NO₂ to 2‐NO₂) might result from the increased hydrophobicity of residues 41 and 124 (proximal to FMN) and conformational changes in residues 70 and 124.     

Structure–Activity Relationships of Benzenesulfonamide‐Based Inhibitors towards Carbonic Anhydrase Isoform Specificity

Carbonic anhydrases (CAs) are implicated in a wide range of diseases, including the upregulation of isoforms CA IX and XII in many aggressive cancers. However, effective inhibition of disease‐implicated CAs should minimally affect the ubiquitously expressed isoforms, including CA I and II, to improve directed distribution of the inhibitors to the cancer‐associated isoforms and reduce side effects. Four benzenesulfonamide‐based inhibitors were synthesized by using the tail approach and displayed nanomolar affinities for several CA isoforms. The crystal structures of the inhibitors bound to a CA IX mimic and CA II are presented. Further in silico modeling was performed with the inhibitors docked into CA I and XII to identify residues that contributed to or hindered their binding interactions. These structural studies demonstrated that active‐site residues lining the hydrophobic pocket, especially positions 92 and 131, dictate the positional binding and affinity of inhibitors, whereas the tail groups modulate CA isoform specificity. Geometry optimizations were performed on each ligand in the crystal structures and showed that the energetic penalties of the inhibitor conformations were negligible compared to the gains from active‐site interactions. These studies further our understanding of obtaining isoform specificity when designing small molecule CA inhibitors.     

Single‐Molecule Kinetics of an Enzyme in the Presence of Multiple Substrates

Herein, the catalytic activity of a single enzyme in the presence of multiple substrates is studied. Three different mechanisms of bisubstrate binding, namely, ordered sequential, random sequential and ping‐pong nonsequential pathway, are broadly discussed. By means of the chemical master equation approach, exact expressions for the waiting‐time distributions, the mean turnover time and the randomness parameter as a function of the substrate concentration, such that both concentrations are fixed, but one of them is changed quasi‐statically are obtained. The randomness parameter is not equal to unity at intermediate to high substrate concentrations, which indicates the presence of multiple rate‐limiting steps in the reaction pathway in all three modes of bisubstrate binding. This arises due to transitions between the free enzyme and the enzyme–substrate complexes that occur on comparable timescales. Such turnover statistics of the single enzyme can also distinguish between the different types of bisubstrate binding mechanisms.     

Expansion of Access Tunnels and Active‐Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein–solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady‐state kinetics assays, molecular dynamics simulations, and time‐resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme–solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K_0.5 and increasing k_cat, or caused enzyme inactivation by promoting substrate inhibition and decreasing k_cat. The induced activation and inhibition of the enzymes correlated with expansion of the active‐site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active‐sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein–solvent pair and effective cosolvent concentration.     

Structure–Activity Relationships and Molecular Docking of Novel Dihydropyrimidine‐Based Mitotic Eg5 Inhibitors

Dihydropyrimidine‐based compounds belong to the first discovered inhibitors of the human mitotic kinesin Eg5. Although they are used by many research groups as model compounds for chemical genetics, considerably less emphasis has been placed on the improvement of this type of inhibitor, with the exception of two recent studies. Dihydropyrimidines can be divided into class I (analogues that bind in the S configuration) and class II type inhibitors, which bind in the R configuration. Herein we report the synthesis and optimization of novel class II type dihydropyrimidines using a combination of in vitro and docking techniques.     

Redesign of the Phosphate Binding Site of L‐Rhamnulose‐ 1‐Phosphate Aldolase towards a Dihydroxyacetone Dependent Aldolase

The aldol addition of unphosphorylated dihydroxyacetone (DHA) to aldehydes catalyzed by L ‐rhamnulose‐1‐phosphate aldolase (RhuA), a dihydroxyacetone phosphate‐dependent aldolase, is reported. Moreover, a single point mutation in the phosphate binding site of the RhuA wild type, that is, substitution of aspartate for asparagine at position N29, increased by 3‐fold the of aldol addition reactions of DHA to other aldehyde acceptors rather than the natural L ‐lactaldehyde. The RhuA N29D mutant modified the optimum enzyme design for the natural substrate and changed its catalytic properties making the aldolase more versatile to other aldol additions of DHA.     

High‐Throughput Bead‐Based Identification of Structure‐Switching Aptamer Beacons

We describe a new platform to identify structure‐switching DNA beacon aptamers, which detect small molecules in a specific manner. By clonally amplifying a DNA library designed to fluoresce in response to binding events onto microbeads, aptamer beacons can be selected by stringent fluorescence‐assisted sorting. We validated this method by isolating known and novel anti‐steroid aptamers from two separate DNA libraries that were structurally enriched with three‐way junctions. Importantly, aptamers were retrieved in only a few (three) rounds of selection by this approach and did not require further optimization, significantly streamlining the process of beacon development.     

Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

A meso‐diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D ‐amino acids other than its native substrate. Site‐directed mutagenesis similar to that carried out on the residues mutated by Vedha‐Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D ‐amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso‐diaminopimelate (meso‐DAP), D ‐leucine (D ‐leu), and 4‐methyl‐2‐oxopentanoic acid (MOPA) were solved. meso‐DAP was found in an area outside the catalytic cavity; this suggested a possible two‐step substrate‐binding mechanism for meso‐DAP. D ‐leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso‐diaminopimelate dehydrogenases.     

Structure‐Dependent Binding of Arylimidamides to the DNA Minor Groove

Heterocyclic diamidines are strong DNA minor‐groove binders and have excellent antiparasitic activity. To extend the biological activity of these compounds, a series of arylimidamides (AIAs) analogues, which have better uptake properties in Leishmania and Trypanosoma cruizi than diamidines, was prepared. The binding of the AIAs to DNA was investigated by T_m, fluorescence displacement titration, circular dichroism, DNase I footprinting, biosensor surface plasmon resonance, X‐ray crystallography and molecular modeling. These compounds form 1:1 complexes with AT sequences in the DNA minor groove, and the binding strength varies with substituent size, charge and polarity. These substituent‐dependent structure and properties provide a SAR that can be used to estimate K values for binding to DNA in this series. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for AIAs.     

Single‐Molecule Unzipping Force Analysis of HU–DNA Complexes

The genome of bacteria is organized and compacted by the action of nucleoid‐associated proteins. These proteins are often present in tens of thousands of copies and bind with low specificity along the genome. DNA‐bound proteins thus potentially act as roadblocks to the progression of machinery that moves along the DNA. In this study, we have investigated the effect of histone‐like protein from strain U93 (HU), one of the key proteins involved in shaping the bacterial nucleoid, on DNA helix stability by mechanically unzipping single dsDNA molecules. Our study demonstrates that individually bound HU proteins have no observable effect on DNA helix stability, whereas HU proteins bound side‐by‐side within filaments increase DNA helix stability. As the stabilizing effect is small compared to the power of DNA‐based motor enzymes, our results suggest that HU alone does not provide substantial hindrance to the motor's progression in vivo.     

Molecular Basis for the Stereoselective Ammoniolysis of N‐Alkyl Aziridine‐2‐Carboxylates Catalyzed by Candida antarctica Lipase B

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N‐alkyl aziridine‐2‐carboxylates in tBuOH saturated with ammonia and yielded the (2S)‐aziridine‐2‐carboxamide and unreacted (2R)‐aziridine‐2‐carboxylate. Varying the N‐1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1′R)‐1‐phenylethyl substituent reacted approximately ten times faster than substrates with a (1′S)‐1‐phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5–7) while substrates with either a (1′R)‐ or (1′S)‐1‐phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity—binding of the slow enantiomer by an exchange‐of‐substituents orientation—could not account for the experimental results. However, modeling an umbrella‐like‐inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1′S)‐1‐phenylethyl substituent and Thr138 and Ile189 in the acyl‐binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella‐like‐inversion orientation can contribute to enantioselectivity in lipases.     

Structure–Activity Relationships of SSAO/VAP‐1 Arylalkylamine‐Based Substrates

SSAO/VAP‐1 substrates may be valuable for the treatment or prevention of diabetes mellitus, as they show insulin‐mimetic properties. This review highlights the importance of studying the relevant steric and electronic features in the development of new ligands with better SSAO/VAP‐1 recognition, enhanced selectivity over other amine oxidases, and improved metabolic behavior.

     

Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.     

Designed RNAs with Two Peptide‐Binding Units as Artificial Templates for Native Chemical Ligation of RNA‐Binding Peptides

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient “RNA‐polypeptide (RNP) world,” which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide‐binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self‐dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.     

Novel Insights into Structure–Activity Relationships of N‐Terminally Modified PACE4 Inhibitors

PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi‐Leu (ML) peptide, an octapeptide with the sequence Ac‐LLLLRVKR‐NH₂. Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N‐terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N‐terminal extension but by the protection of both ends with the d ‐Leu residue and 4‐amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile.     

Computational Study of the Lipase‐Mediated Desymmetrisation of 2‐Substituted‐Propane‐1,3‐Diamines

The enantioselectivity displayed by the lipase from Pseudomonas cepacia towards a wide range of prochiral 2‐substituted‐propane‐1,3‐diamines was studied by means of molecular dynamics simulations (MDS). In all cases the enzyme allows the recovery of the corresponding amino carbamates of R configuration. However, the enantioselectivity is only synthetically useful if no ortho substituent is present and the aromatic ring is directly bonded to the 2‐carbon of the 1,3‐diamine core. Analysis of the MDS trajectories revealed that the homologation of 2‐aryl substituents by means of a methylene group lowers enantioselectivity by alleviating the conformational tension of the slow‐reacting orientations due to unfavourable intramolecular contacts between the ortho carbons of the aryl group and the nucleophilic nitrogen, as well as between the chiral carbon and the oxyanion. Additionally, the relative solvent accessible surfaces of the atoms of the aryl ring nicely correlate with the effect of the location of the substituent on enantioselectivity.     

Benzaldehyde Lyase‐Catalyzed Direct Amidation of Aldehydes with Nitroso Compounds

Benzaldehyde lyase from the Pseudomonas fluorescens catalyzes the reaction of aromatic aldehydes with nitroso compounds and furnishes N‐arylhydroxamic acids in high yields. Aromatic aldehydes and benzoins are converted into enamine‐carbanion‐like intermediates prior to their reaction with nitroso compounds. The kinetic resolution of rac‐2‐hydroxy‐1,2‐diphenylethanones furnished (S)‐benzoins and arylhydroxamic acids with high enantioselectivities and conversions.