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Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii , we observed that Ses- Tw TPS1-1 and Ses- Tw TPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses- Tw TPS1-2, Ses- Tw TPS3, and Ses- Tw TPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E , E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses- Tw TPS1-1:V199I and Ses- Tw TPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio. [ABSTRACT FROM AUTHOR]

• Identification of glycosyltransferases from Bacillus subtilis converting HQNO. • Kinetic parameters of YjiC, YdhE and Yojk towards HQNO and UDP-glucose. • Identification of glucosylated plant and Pseudomona s derived antimicrobials. • Comparison of NDP-glucose and aglycon promiscuities. Glycosylation of natural and synthetic products can alter the physical, chemical and pharmacological properties of the aglycon. Conversion of 2-heptyl-1-hydroxyquinolin-4-one (HQNO), a potent respiratory inhibitor produced by Pseudomonas aeruginosa , to the less toxic 2-heptyl-1-(β-D-glucopyranosydyl)-quinolin-4-one, was recently demonstrated for Bacillus subtilis strain 168. In this study, we compared the genomes of several Bacillus spp. to identify candidate enzymes for HQNO glucosylation. All three (putative) UDP-glycosyltransferases (GT) of B. subtili s 168 tested, YjiC, YdhE and YojK, were capable of HQNO glucosylation, with YjiC showing the highest turnover rate (k cat) of 4.6 s−1, and YdhE exhibiting the lowest K m value for HQNO of 9.1 μM. All three GT predominantly utilized UDP-glucose, but YdhE was similarly active with TDP-glucose. Among the aglycons tested, HQNO was the preferred substrate of all three GT, but they also showed activities toward the P. aeruginosa exoproducts pyocyanin, 2-heptyl-3-hydroxyquinolin-4(1 H)-one (the Pseudomonas quinolone signal) and 2,4-dihydroxyquinoline, the plant derived antimicrobials vanillin and quercetin, and the macrolide antibiotic tylosin A. Our results underline the promiscuity and substrate flexibility of YjiC, YdhE and YojK, and suggest a physiological role in natural toxin resistance of B. subtilis. Especially YdhE appears to be an attractive biocatalyst for the glycoengineering of natural products. [ABSTRACT FROM AUTHOR]

CotA laccases are multicopper oxidases known for promiscuously oxidizing a broad range of substrates. However, studying substrate promiscuity is limited by the complexity of electron transfer (ET) between substrates and laccases. Here, a systematic analysis of factors affecting ET including electron donor acceptor coupling (Η DA), driving force (ΔG) and reorganization energy (λ) was done. Catalysis rates of syringic acid (SA), syringaldehyde (SAD) and acetosyringone (AS) (kcat(SAD) > kcat(SA) > kcat(AS)) are not entirely dependent on the ability to form phenol radicals indicated by ΔG and λ calculated by Density Functional Theory (SA < SAD ≈ AS). In determined CotA/SA and CotA/SAD structures, SA and SAD bound at 3.9 and 3.7 Å away from T1 Cu coordinating His419 ensuring a similar Η DA. Abilities of substrate to form phenol radicals could mainly account for difference between kcat(SAD) and kcat(SA). Furthermore, substrate pocket is solvent exposed at the para site of substrate's phenol hydroxyl, which would destabilize binding of AS in the same orientation and position resulting in low kcat. Our results indicated shallow partially covered binding site with propensity of amino acids distribution might help CotA discriminate lignin-phenol derivatives. These findings give new insights for developing specific catalysts for industrial application. [ABSTRACT FROM AUTHOR]

CotA laccases are multicopper oxidases known for promiscuously oxidizing a broad range of substrates. However, studying substrate promiscuity is limited by the complexity of electron transfer (ET) between substrates and laccases. Here, a systematic analysis of factors affecting ET including electron donor acceptor coupling (Η DA), driving force (ΔG) and reorganization energy (λ) was done. Catalysis rates of syringic acid (SA), syringaldehyde (SAD) and acetosyringone (AS) (kcat(SAD) > kcat(SA) > kcat(AS)) are not entirely dependent on the ability to form phenol radicals indicated by ΔG and λ calculated by Density Functional Theory (SA < SAD ≈ AS). In determined CotA/SA and CotA/SAD structures, SA and SAD bound at 3.9 and 3.7 Å away from T1 Cu coordinating His419 ensuring a similar Η DA. Abilities of substrate to form phenol radicals could mainly account for difference between kcat(SAD) and kcat(SA). Furthermore, substrate pocket is solvent exposed at the para site of substrate's phenol hydroxyl, which would destabilize binding of AS in the same orientation and position resulting in low kcat. Our results indicated shallow partially covered binding site with propensity of amino acids distribution might help CotA discriminate lignin-phenol derivatives. These findings give new insights for developing specific catalysts for industrial application. [ABSTRACT FROM AUTHOR]

The substrate specificity of enzymes is bound to be imperfect, because of unavoidable physicochemical limits. In extant metabolic enzymes, furthermore, such limits are seldom approached, suggesting that the degree of specificity of these enzymes, on average, is much lower than could be attained. During biological evolution, the activity of a single enzyme with available alternative substrates may be preserved to a significant or even substantial level for different reasons – for example when the alternative reaction contributes to fitness, or when its undesirable products are nevertheless dispatched by metabolite repair enzymes. In turn, the widespread occurrence of promiscuous reactions is a consistent source of metabolic 'messiness', from which both liabilities and opportunities ensue in the evolution of metabolic systems. Highlights Substrate specificity cannot be absolute and is inherently limited. The maximum capacity to discriminate between alternative substrates can be relatively low, and in any case it is seldom approached owing to evolutionary constraints. In some enzymes of primary metabolism, substrate promiscuity is favored, although this may interfere with high flux and efficient regulation. In other cases, enzymes acting on alternative substrates generate toxic or useless products; however, these can be destroyed or recycled by repair enzymes. The limited substrate specificity of enzymes often results in the production of non-standard metabolites which contribute to the complexity of the metabolome. Substrate promiscuity helps to fuel an 'underground' network of reactions which may represent a basis for further evolution and diversification of metabolism. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 (CYP3A4) metabolizes a wide range of drugs and toxins. Interactions of CYP3A4 with ligands are difficult to predict due to promiscuity and conformational flexibility. To better understand CYP3A4 conformational responses to ligands we use hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the effect of ligands on nanodisc-embedded CYP3A4. For a subset of CYP3A4-ligand complexes, differences in the low-frequency modes derived by principal component analyses of molecular dynamics trajectories mirrored the HDX-MS results. The effects of ligands are distributed to flexible elements of CYP3A4 between stretches of secondary structure. The largest effects occur in the F- and G-helices, where most ligands increase the flexibility of the F-helix and connecting loops and decrease the flexibility of the C-term of the G-helix. Most ligands affect the E -F-G, C D and H I regions of the protein. Ligand-dependent differences are observed in the A"-A' loop, B C region, E -helix, K-β1 region, proximal loop, and C-term loop. Correlated HDX responses were observed in the C D region and the C-term of the G-helix that were most pronounced for Type II ligands. Collectively, the HDX and molecular dynamics results suggest that CYP3A4 accommodates diverse binding partners by propagating local backbone fluctuations from the binding site onto the flexible regions of the enzyme via long-range interactions that are differentially modulated by ligands. In contrast to the paradigm wherein ligands decrease protein dynamics at their binding site, a wide range of ligands modestly increase CYP3A4 dynamics throughout the protein including effects remote from the active site. Superimposition of average structures of CYP3A4 from GaMD simulations of the ligand-free CYP3A4 (tan), CYP3A4-Azimulin complex (blue), and CYP3A4-Ritonavir complex (pink) illustrating the differences in the packing of the F-helix and E -F loop against the E-helix. MD and H/DX mass spectrometry confirm regions uniquely or universally affected by various ligands. [Display omitted] • CYP3A4 is a highly promiscuous detoxication enzyme. • H/DX MS reveals CYP3A4 ligands cause globally distributed changes in dynamics. • Results are supported by MD simulations and principle component analysis. • Ligands induce an increase in local dynamics far from the binding site. [ABSTRACT FROM AUTHOR]

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) represents the main subtype of the NPP family of nucleotide hydrolyzing enzymes. The ecto-enzyme hydrolyzes structurally diverse substrates and has recently been proposed as a drug target for immuno-oncology. To get more insights into the nature of the promiscuity of NPP1, we investigated its substrate preferences employing a broad range of natural nucleotides including ATP, UTP, diadenosine tetraphosphate (AP 4 A), cAMP, and cyclic guanosine-(2′,5′)-monophosphate-adenosine-(3″,5″)-monophosphate (2′,3″-cGAMP), as well as the artificial substrate p -nitrophenyl 5′-thymidine monophosphate ( p -Nph-5′-TMP). Despite their diverse structures, all substrates were converted to nucleoside 5′-monophosphates; 2′,3″-cGAMP yielded exclusively the nucleoside 5′-monophosphates AMP and GMP. In contrast, 3′,3″-bridged cyclic dinucleotides were not hydrolyzed. ATP was the most efficiently hydrolyzed substrate of NPP1, followed by AP 4 A and 2′,3″-cGAMP. UTP, cAMP and p -Nph-5′-TMP were much poorer substrates. A homology model of the human NPP1 was built based on the X-ray structure of its mouse orthologue. Docking studies were performed based on previously published mutagenesis data to rationalize the interactions of the different substrates and to explain the enzyme's preferences. The results provide an improved understanding of the interactions of NPP1 with its diverse substrates and will contribute to the validation of NPP1 as a drug target. [ABSTRACT FROM AUTHOR]

• Functional characterization of five different UGDH isoforms in G. uralensis. • Enzyme kinetics study of the isoforms. • Substrate promiscuity exploration of the isoforms. • Molecular docking, MD-Simulation, and binding free energy calculation. Uridine diphosphate glucose dehydrogenase (UGDH) has a crucial role in the synthesis of UDP-glucuronic acid (UDPGA), which is the important sugar donor in the glucuronosylation of bioactive triterpene saponins in the traditional Chinese medicine Glycyrrhiza uralensis Fisch. UGDHs usually exist as isoforms in plants, but knowledge of their unique binding sites and patterns remains largely incomplete. Here, we mined five UGDH isoforms in G. uralensis transcriptome that catalyze the oxidation of UDP-glucose (UDPG) to UDPGA and had different catalytic efficiencies. Molecular modeling and molecular dynamics simulation revealed the potential catalytic residues for the catalytic efficiencies of the isoforms. Moreover, the UGDH isoforms exhibited substrate promiscuity by catalyzing two structurally diverse UDPG derivatives, which was further demonstrated by docking studies. This work gives new insights into the UGDH isoforms involved in the biosynthesis of glucuronides in G. uralensis , and extends the understanding of the versatility of UGDHs. [ABSTRACT FROM AUTHOR]

7-DMATS from Aspergillus fumigatus and SirD from Leptosphaeria maculans catalyse a C 7-prenylation of l -tryptophan and an O -prenylation of l -tyrosine in nature, respectively. SirD was reported to catalyse the C 7-prenylation of l -tryptophan and some derivatives thereof in vitro. We report here the O-prenylation of tyrosine and O - or N -prenylation of its derivatives by 7-DMATS. These results provide experimental evidence for the close relationship of tyrosine O - and tryptophan C 7-prenyltransferases regarding their substrate and catalytic promiscuity. [ABSTRACT FROM AUTHOR]

Abstract: In organic synthesis the Friedel–Crafts alkylation is of eminent importance, as it is a key reaction in many synthetic routes. A general access to enzymatic Friedel–Crafts alkylations would be very beneficial due to the high selectivity of biocatalysts. We used designed polyprenyl phenyl ethers to specifically address this reaction by using squalene hopene cyclases as catalysts. Polycyclic products with aromatic rings constituting important biological active compounds were obtained. Our results demonstrate that squalene hopene cyclases can be utilized for Friedel–Crafts alkylations and reveal the potential of these enzymes for chiral Brønsted acid catalysis. [Copyright &y& Elsevier]

Abstract: The substrate preferences of three acylases – two wild-type enzymes and an evolved variant obtained by directed evolution – which are prototypical enzymes for glutaryl-7-ACA acylase and cephalosporin C acylase subfamilies, have been investigated. A preliminary screening of enzymes’ performances on a large set of substrates has been carried out by a colorimetric assay performed in 96-well plates and by a pH-Stat monitoring the hydrolytic activities. Subsequently, kinetic data for selected substrates have been determined, thus elucidating the substrate preference of members of glutaryl-7-ACA acylase vs. cephalosporin C acylase subfamilies. These achievements pave the way to the ability of choosing the best enzyme for the hydrolysis of different compounds of industrial importance. [Copyright &y& Elsevier]

Abstract: In this study, the transglucosylation potential of six sucrose phosphorylase (SP) enzymes has been compared using eighty putative acceptors from different structural classes. To increase the solubility of hydrophobic acceptors, the addition of various co-solvents was first evaluated. All enzymes were found to retain at least 50% of their activity in 25% dimethylsulfoxide, with the enzymes from Bifidobacterium adolescentis and Streptococcus mutans being the most stable. Screening of the enzymes’ specificity then revealed that the vast majority of acceptors are transglucosylated very slowly by SP, at a rate that is comparable to the contaminating hydrolytic reaction. The enzyme from S. mutans displayed the narrowest acceptor specificity and the one from Leuconostoc mesenteroides NRRL B1355 the broadest. However, high activity could only be detected on l-sorbose and l-arabinose, besides the native acceptors d-fructose and phosphate. Improving the affinity for alternative acceptors by means of enzyme engineering will, therefore, be a major challenge for the commercial exploitation of the transglucosylation potential of sucrose phosphorylase. [Copyright &y& Elsevier]

Abstract: Most cardenolides used in the therapy of cardiac insufficiency are 5β-configured and thus the stereo-specific reduction of the Δ4,5-double bond of a steroid precursor is a crucial step in their biosynthesis. This step is thought to be catalysed by progesterone 5β-reductases. We report here on the isolation of 11 progesterone 5β-reductase (P5βR) orthologues from 5β-cardenolide-free and 5β-cardenolide-producing plant species belonging to five different angiosperm orders (Brassicales, Gentianales, Lamiales, Malvales and Solanales). Amino acid sequences of the P5βR described here were highly conserved. They all contain certain motifs qualifying them as members of a class of stereo-selective enone reductases capable of reducing activated Cbonds by a 1,4-addition mechanism. Protein modeling revealed seven conserved amino acids in the substrate-binding/catalytic site of these enzymes which are all supposed to exhibit low substrate specificity. Eight P5βR genes isolated were expressed in Escherichia coli. Recombinant enzymes reduced progesterone stereo-specifically to 5β-pregane-3,20-dione. The progesterone 5β-reductases from Digitalis canariensis and Arabidopsis thaliana reduced activated Cbonds of molecules much smaller than progesterone. The specific role of progesterone 5β-reductases of P5βRs in cardenolide metabolism is challenged because this class of enone reductases is widespread in higher plants, and they accept a wide range of enone substrates. [ABSTRACT FROM AUTHOR]