Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k_cat/K_m values >10,000 M^?1 s^?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k_cat/K_m values exhibited against these canonical substrates are well under 10⁵ M^?1 s^?1. By contrast, several Nudix enzymes show much larger k_cat/K_m values (in the range of 10⁵ to >10⁷ M^?1 s^?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

PURIFICATION AND PHYSICOKINETIC CHARACTERIZATION OF A GLUCONOLACTONE INHIBITION-INSENSITIVE β-GLUCOSIDASE FROM Andrographis paniculata NEES. LEAF

A gluconolactone inhibition-insensitive β-glucosidase from Andrographis paniculata (Acanthaceae) leaves has been isolated, homogeneity purified, and characterized for its physicokinetic properties. The purified enzyme appeared to be a monomeric structure with native molecular weight about 60 kD. The enzyme exhibited optimum pH 5.5 and pI 4.0, meso-thermostability and high temperature optimum (55°C) for catalytic activity, with activation energy of 6.8 kcal Mol^?1. The substrate saturation kinetics studies of the enzyme revealed a Michaelis–Menten constant (K_m) of 0.25 mM for pNPG and catalytic efficiency (K_cat/K_m) of 52,400 M ^?1 s^?1, respectively. Substrate specificity of the enzyme was restricted to β-linked gluco-, manno- and fuco-conjugates. The gluconolactone inhibition insensitivity was evident from its very low inhibition at millimolar inhibitor concentrations. Interestingly, the enzyme showed geraniol transglucosylating activity with pNPG as glucosyl donor but not with cellobiose. The catalytic activity of the enzyme has been reported to be novel with respect to its activity and preferences from a medicinal plant resource.     

Phytaspase,a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine‐dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase‐specific proteolytic activity. Nevertheless, plants do display caspase‐like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase‐like proteases. Here, we report the identification and characterisation of a novel PCD‐related subtilisin‐like protease from tobacco and rice named phytaspase (plant aspartate‐specific protease) that possesses caspase specificity distinct from that of other known caspase‐like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD‐related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re‐imported into the cell during PCD providing insights into how phytaspase operates.     

A broad specificity nucleoside kinase from Thermoplasma acidophilum

The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1‐phosphofructokinases, 6‐phosphofructo‐2‐kinase, inosine/guanosine kinases, fructokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase‐like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB‐like family substrates; activity was not observed for ribose, fructose‐1‐phosphate, or fructose‐6‐phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49, and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent K_M for guanosine of 0.21 μM and catalytic efficiency of 345,000 M^?1s^?1. These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK is a member of a new sub‐family with broad nucleoside specificities. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

Conversion of the catalytic specificity of alanine racemase to a -amino acid aminotransferase activity by a double active-site mutation

Alanine racemase depending on pyridoxal 5′-phosphate catalyzes the interconversion between - and -alanine. The enzyme from Bacillus stearothermophilus catalyzes the transamination as a side reaction with both substrates once per 3×10⁷ times of the racemization. In this work, we studied the effects of the mutation of Arg219, and that of Arg219 and Tyr265 on the catalysis of Bacillus alanine racemase. Arg219 interacting with pyridinium nitrogen of the cofactor is conserved in all alanine racemases. The corresponding residue of aminotransferases is an acidic residue, such as glutamate or aspartate. Mutation of Arg219 to a glutamyl residue resulted in a 5.4-fold increase in the forward half transamination activity with -alanine and a 10³-fold decrease in the racemase activity. The double mutation, Arg219→Glu and Tyr265→Ala, completely abolished the racemase activity and increased the forward half transaminase activity 6.6-fold. Arg219 is one of the structural determinants of the catalytic specificity of the alanine racemase.     

Synergetic DNA‐Cleaving Activities of the Metal Complexes of a Polyether‐Tethered Pyrrole‐polyamide Dimer

A simple polyether‐tethered pyrrole‐polyamide dimer 1 was synthesized in 50% yield from the reaction of 2,2,2‐trichloro‐1‐(1‐methyl‐4‐nitro‐1H‐pyrrol‐2‐yl)ethanone with 2,2′‐[1,2‐ethanediylbis(oxy)]bisethanamine, and fully characterized on the basis of ¹H‐ and ¹³C‐NMR, MS, HR‐MS, and IR data. Agarose gel‐electrophoresis study of the cleavage of plasmid pBR322 DNA by the complexes of compound 1 with seven metal ions indicated that most of the metal complexes were capable of efficiently cleaving DNA at pH 7.0 and 37°. Among them, the Cu^II complex exhibited the highest activity, with the maximal catalytic rate constant k_max and Michaelis constant K_M being 5.61 h^?1 and 7.30 mM , respectively. Spectroscopic, ESI‐MS, ethidium‐bromide (EB) displacement, and viscosity experiments indicated that compound 1 could form a 1 : 1 complex with Cu^II ion, and that this complex showed moderate binding affinity toward calf‐thymus DNA.     

Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO₃^? uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO₃^? (NO₃^?-grown), 0.25 mM NO₃^? (N-limited) or 10 mM NO₃^? followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO₃^?, although there were variations in the uptake V_max of both isotherms. The rate of uptake via isotherm 0 ([NO₃^?]<1.1 mM) in N-limited cells was higher than that of either NO₃^?-grown or N-starved cells. In contrast, NO₃^?-grown and N-limited cells exhibited comparable V_max values when supplied with 1.1 to 1.8 mM NO₃^? (isotherm 1). When supplied with 1.6 mM NO₃^?, both N-limited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO₃^?-grown cells were supplied with 1.6 mM NO₃^?, or when N-limited and N-starved cells were supplied with 0.6 mM NO₃^?. The ‘inducible’ aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg l^?1), but not by actinomycin D (5 mg l^?1), thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO₃^? for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17–73 kDa, pI ca 5.0 to 7.1) unique to the high NO₃^? treatment. We propose that the ‘inducible’ portion of NO₃^? uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.     

The X-ray crystallographic structure and specificity profile of HAD superfamily phosphohydrolase BT1666: comparison of paralogous functions in B. thetaiotaomicron

Analysis of the haloalkanoate dehalogenase superfamily (HADSF) has uncovered homologues occurring within the same organism that are found to possess broad, overlapping substrate specificities, and low catalytic efficiencies. Here we compare the HADSF phosphatase BT1666 from Bacteroides thetaiotaomicron VPI‐5482 to a homologue with high sequence identity (40%) from the same organism BT4131, a known hexose‐phosphate phosphatase. The goal is to find whether these enzymes represent duplicated versus paralogous activities. The X‐ray crystal structure of BT1666 was determined to 1.82 Å resolution. Superposition of the BT1666 and BT4131 structures revealed a conserved fold and identical active sites suggestive of a common physiological substrate. The steady‐state kinetic constants for BT1666 were determined for a diverse panel of phosphorylated metabolites to define its substrate specificity profile and overall level of catalytic efficiency. Whereas BT1666 and BT4131 are both promiscuous, their substrate specificity profiles are distinct. The catalytic efficiency of BT1666 (k_cat/K_m = 4.4 × 10²M^?1 s^?1 for the best substrate fructose 1,6‐(bis)phosphate) is an order of magnitude less than that of BT4131 (k_cat/K_m = 6.7 × 10³M^?1 s^?1 for 2‐deoxyglucose 6‐phosphate). The seemingly identical active‐site structures point to sequence variation outside the active site causing differences in conformational dynamics or subtle catalytic positioning effects that drive the divergence in catalytic efficiency and selectivity. The overlapping substrate profiles may be understood in terms of differential regulation of expression of the two enzymes or a conferred advantage in metabolic housekeeping functions by having a larger range of possible metabolites as substrates. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

Cholecystokinin and tryptic activity in the gut and body of developing Atlantic halibut larvae: evidence for participation in the regulation of protein digestion

At 7 days after first feeding (DAFF), the peptide hormone cholecystokinin (CCK) content (fmol individual^?1) and the tryptic activity [μmol arginine‐methyl‐coumarinyl‐7‐amide (MCA) min^?1 individual^?1] per individual gut of Atlantic halibut Hippoglossus hippoglossus larvae were low: 0·2 ± 0·1 and 0·14 ± 0·10, respectively. Thereafter, both parameters increased with the increase in gut mass and reached 19·67 ± 5·58 and 2·71 ± 0·64 at 26 DAFF, respectively. Due to the small sample size, the dry mass (M_G, mg) of the individual gut could not be determined accurately at 7 DAFF. At 13 DAFF M_G represented 5·5% of whole body dry mass (M_w, mg) while at 26 DAFF it had increased to 23%. The mass specific tryptic activity [μmol MCA min^?1 per mg dry mass (M)] in the gut increased from 2·74 ± 1 ± 98 at 13 DAFF to 5·00 ± 0·78 at 26 DAFF. There was more individual variation in the mass specific CCK content (fmol M^?1) but no significant differences were found, although the data indicated an increase (from 23·38 ± 11·26 at 13 DAFF to 36·27 ± 8·96 fmol M^?1 at 26 DAFF). At 7 DAFF the CCK content of the gut represented c. 2% of the whole body CCK content while it increased to c. 62% of the whole body CCK content at 26 DAFF. This demonstrates that it is necessary to separate neural and gastrointestinal sources of CCK in order to determine its alimentary role in fish larvae. Trypsin activity was only found in the gut compartment. In larvae aged 45 DAFF dietary proteins delivery into the gut by tube‐feeding appeared to stimulate post‐prandial secretion of CCK from the gut as well as stimulate pancreatic trypsin secretion, suggesting that both factors contribute to protein digestion.     

Determination of the Superoxide Dismutase-Like Activity of Cimetidine-Cu(II) Complexes

Using the direct method of pulse radiolysis to determine the superoxide dismutase like activity of copper(II) cimetidine complexes, it was found that the reaction rate constant with O^?₂, k_cat, was (8.5 ± 0.5) × 10⁸ M^?1s^?1 independent of the cimetidine concentrations present in excess of 50–200 μM over the metal. The results suggest that either the 1:1 ligand to metal complex does not catalyze O^?₂ dismutation at a comparable rate to that of the 2:1 complex, or that the stability constant of the last species is much higher than that determined earlier by Kimura el al.,¹ and only the 2:1 species is present in the solutions. With the indirect methods of cytochrome c and NBT for determining the ability of these complexes to catalyze O^?₂ dismutation, these compounds exhibited a much lower SOD activity. and k_cat was determined to be (5.0 ± 0.3) × 10⁶ and (7.± 0.4) × 10¹ M^?1s^?1. respectively using the two assays.     

Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Four insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera, and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization using different colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from bovine chymotrypsin in their primary specificity toward small substrates (like N‐benzoyl‐L‐Tyr p‐nitroanilide) rather than on their preference for large substrates (exemplified by Succynil‐Ala‐Ala‐Pro‐Phe p‐nitroanilide). Chloromethyl ketones (TPCK, N‐ α‐tosyl‐L‐Phe chloromethyl ketone and Z‐GGF‐CK, N‐ carbobenzoxy‐Gly‐Gly‐Phe‐CK) inactivated all chymotrypsins tested. Inactivation rates follow apparent first‐order kinetics with variable second order rates (TPCK, 42 to 130 M^?1 s^?1; Z‐GGF‐CK, 150 to 450 M^?1 s^?1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M^?1 s^?1; Z‐GGF‐CK, 6.1 M^?1 s^?1). Homology modelling and sequence alignment showed that in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa value. This is proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed to be an adaptation to the presence of dietary ketones. © 2009 Wiley Periodicals, Inc.     

Polyphenol Characterization,Antioxidant and Skin Whitening Properties of Alnus cordata Stem Bark

In this study, we investigated the phenolic composition of the crude extract (MeOH 80 %) of Alnus cordata (Loisel .) Duby stem bark (ACE) and its antioxidant and skin whitening properties. RP‐LC‐DAD analysis showed a high content of hydroxycinnamic acids (47.64 %), flavanones (26.74 %) and diarylheptanoids (17.69 %). Furthermore, ACE exhibited a dose‐dependent antioxidant and free‐radical scavenging activity, expressed as half‐maximal inhibitory concentration (IC₅₀): Oxygen radical absorbance capacity (ORAC, IC₅₀ 1.78 μg mL^?1)>Trolox equivalent antioxidant capacity (TEAC, IC₅₀ 3.47 μg mL^?1)>2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH, IC₅₀ 5.83 μg mL^?1)>β‐carotene bleaching (IC₅₀ 11.58 μg mL^?1)>Ferric reducing antioxidant power (FRAP, IC₅₀ 17.28 μg mL^?1). Moreover, ACE was able to inhibit in vitro tyrosinase activity (IC₅₀ 77.44 μg mL^?1), l ‐DOPA auto‐oxidation (IC₅₀ 39.58 μg mL^?1) and in an in vivo model it exhibited bleaching effects on the pigmentation of zebrafish embryos (72 h post fertilization) without affecting their development and survival. In conclusion, results show that A. cordata stem bark may be considered a potential source of agents for the treatment of skin disorders due to its bleaching properties and favorable safety profiles, associated to a good antioxidant power.     

Characterization of cross‐linked immobilized arylesterase from Gluconobacter oxydans 621H with activity toward cephalosporin C and 7‐aminocephalosporanic acid

Cross‐linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross‐linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7‐aminocephalosporanic acid. The CLEAs had a Kcat/K_M of 0.9 M^?1/S^?1 for 7‐ACA (7‐aminocephalosporanic acid) and 0.1 M^?1/S^?1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis–Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36–42, 2016     

Screening,partial purification and characterization of the hyper-thermophilic lipase produced by a new isolate of Bacillus subtilis LP2

Abstract

Lipases are one of the most important catalysts for several industries such as detergent, dairy, and textile industry due to their bio-catalytic ability in aqueous and non-aqueous media. Stability to extreme conditions is an important property since it makes enzymes suitable to several industrial processes. In this study, lipase producing soil bacteria were screened and identified with 16S rDNA sequencing. A new hyper-thermophilic lipase named as Bacillus subtilis LP2 isolate was partially purified by ammonium sulphate precipitation with 17.8-fold purification and 583?U/mg specific activity. Maximum activity was exhibited at pH 7 and 80?°C with the substrate tween 80?K_M and V_max values were calculated as 18.3?mM and 680?U/mg with a catalytic efficiency (k_cat/K_M) of 307?s^?1M^?1. These results indicate that lipase from Bacillus subtilis LP2 can be a valuable candidate for industrial applications such as organic synthesis and fats and oils industry due to their efficient catalysis in higher temperatures.     

Comprehensive Characterisation of n‐Alkylresorcinols and Other Lipid Constituents of Mercurialis tomentosa L. from Alicante,Spain

Mercurialis tomentosa L. has been used in Spanish ethnomedicine. In the present study the first phytochemical characterisation of a lipid fraction from M. tomentosa was performed. The CHCl₃ extraction of aerial parts from M. tomentosa and GC/MS investigations revealed the occurrence of cuticular lipid and wax constituents, like long chain n‐alcohols and n‐aldehydes (C₂₂ – C₃₀), besides several aromatic constituents, i.e., phenylpropanoids and n‐alkylresorcinols. The latter were further purified by CC and analysed by LC/MSⁿ. In contrast to other Mercurialis species, i.e., M. annua, M. perennis, which exclusively contain 5‐n‐alkylresorcinols ( 1a  –  j , C_n), mainly 5‐n‐alkyl‐2‐methylresorcinols ( 2a  –  j , C_n*) with side chain lengths of C₁₅ – C₂₅ were found in M. tomentosa, in addition to 1a  –  j . Thus, the latter compounds may be utilised for analytical characterisation and authentication of M. tomentosa based on fingerprinting methods. For structure elucidation a novel facile total synthesis of one representative 5‐n‐alkyl‐2‐methylresorcinol homologue ( 2d , C₁₉*) was developed, starting with a Grignard reaction from a substituted benzoic acid chloride ( 19 ). The compound obtained by synthesis was identical to the natural product 2d in terms of its chromatographic and spectroscopic features. Futhermore, 2d exhibited satisfactory DPPH free radical scavenging activity (IC₅₀ = 37.8 μm ) when compared to trolox (IC₅₀ = 21.0 μm ), corroborating the antioxidant features of these amphipathic molecules.     

Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-d-glucitol

Purified recombinant sorbose dehydrogenase from Sinorhizobium sp. 97507 exhibited high reactivity for 1,5-anhydro-d-glucitol (1,5-AG) and l-sorbose, but little activity for the other sugars or sugar alcohols tested. Kinetic analysis revealed that its catalytic efficiency (k_cat/K_m) for l-sorbose and 1,5-AG is 1.8 × 10² and 1.5 × 10² s^?1·M^?1, respectively.     

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K _m than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.     

Structure‐kinetic relationship of carbapenem antibacterials permeating through E. coli OmpC porin

The emergence of Gram‐negative “superbugs” exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram‐negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure‐translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC‐ E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC‐expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k_on ≈ 2 × 10⁴ M^?1 s^?1). Zwitterionic compounds with highly polar groups attached to the penem‐2 ring, including panipenem, imipenem and doripenem exhibit faster k_on (>10⁴ M^?1 s^?1), while meropenem and biapenem with fewer exposed polar groups exhibit slower k_on (～5 × 10³ M^?1 s^?1). Tebipenem pivoxil and razupenem exhibit ～13‐fold slower k_on (～1.5 × 10³ M^?1 s^?1) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure‐kinetic relationships of carbapenem translocation are governed by H‐bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups). Proteins 2014; 82:2998–3012. © 2014 Wiley Periodicals, Inc.