New enzymes for peptide biosynthesis in microorganisms.

Peptides, biologically occurring oligomers of amino acids linked by amide bonds, are essential for living organisms. Many peptides isolated as natural products have biological functions such as antimicrobial, antivirus and insecticidal activities. Peptides often possess structural features or modifications not found in proteins, including the presence of nonproteinogenic amino acids, macrocyclic ring formation, heterocyclization, N-methylation and decoration by sugars or acyl groups. Nature employs various strategies to increase the structural diversity of peptides. Enzymes that modify peptides to yield mature natural products are of great interest for discovering new enzyme chemistry and are important for medicinal chemistry applications. We have discovered novel peptide modifying enzymes and have identified: (i) a new class of amide bond forming-enzymes; (ii) a pathway to biosynthesize a carbonylmethylene-containing pseudodipeptide structure; and (iii) two distinct peptide epimerases. In this review, an overview of our findings on peptide modifying enzymes is presented.

Identification and characterization of the 4-epimerase AglW from the archaeon Methanococcus maripaludis.

Archaea are ubiquitous single-cell microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis, a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with N-linked tetrasaccharides that are essential for the proper assembly and function of archaella. The aglW gene, encoding the putative 4-epimerase AglW, plays a key role in the synthesis of the tetrasaccharide. The goal of our work was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop assays to determine its substrate specificity and properties. We carried out assays using UDP-Galactose, UDP-Glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and N-acetylglucosamine/N-acetylgalactosamine-diphosphate - lipid as substrates, coupled with specific glycosyltransferases. We showed that AglW has a broad specificity towards UDP-sugars and that Tyr151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The glycosyltransferase-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases.

Isolation and characterization of racemase from Ensifer sp. 23-3 that acts on α-aminolactams and α-amino acid amides.

Limited information is available on α-amino-ε-caprolactam (ACL) racemase (ACLR), a pyridoxal 5'-phosphate-dependent enzyme that acts on ACL and α-amino acid amides. In the present study, eight bacterial strains with the ability to racemize α-amino-ε-caprolactam were isolated and one of them was identified as Ensifer sp. strain 23-3. The gene for ACLR from Ensifer sp. 23-3 was cloned and expressed in Escherichia coli. The recombinant ACLR was then purified to homogeneity from the E. coli transformant harboring the ACLR gene from Ensifer sp. 23-3, and its properties were characterized. This enzyme acted not only on ACL but also on α-amino-δ-valerolactam, α-amino-ω-octalactam, α-aminobutyric acid amide, and alanine amide.

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from Citreicella sp. SE45.

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS-polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including L-alanine amide and L-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K m and V max values for D- and L-ACL were 5.3 and 2.17 mM, and 769 and 558 µmol/min.mg protein, respectively. Moreover, the turn over number (K cat) and catalytic efficiency (K cat/K m ) of purified ACL racemase from Citreicella sp. SE45 using L-ACL as a substrate were 465 S-1 and 214 S-1mM-1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.

Purification and characterization of d-allulose 3-epimerase derived from Arthrobacter globiformis M30, a GRAS microorganism.

An enzyme that catalyzes C-3 epimerization between d-fructose and d-allulose was found in Arthrobacter globiformis strain M30. Arthrobacter species have long been used in the food industry and are well-known for their high degree of safety. The enzyme was purified by ion exchange and hydrophobic interaction chromatographies and characterized as a d-allulose 3-epimerase (d-AE). The molecular weight of the purified enzyme was estimated to be 128 kDa with four identical subunits. The enzyme showed maximal activity and thermostability in the presence of Mg2+. The optimal pH and temperature for enzymatic activity were 7.0-8.0 and 70°C, respectively. The enzyme was immobilized to ion exchange resin whereupon it was stable for longer periods than the free enzyme when stored at below 10°C. In the column reaction, the enzyme activity also maintained stability for more than 4 months. Under these conditions, 215 kg of d-allulose produced per liter immobilized enzyme, and this was the highest production yield of d-allulose reported so far. These highly stable properties suggest that this enzyme represents an ideal candidate for the industrial production of d-allulose.

Food-Grade Expression of d-Psicose 3-Epimerase with Tandem Repeat Genes in Bacillus subtilis.

An integrative food-grade expression system with tandem repeat target genes was constructed for the expression of d-psicose 3-epimerase (DPEase; EC 5.1.3.30). The DPEase gene fused with the P43 promoter constituted an independent monomeric expression cassette. Multimers of the expression cassette were constructed in vitro using the isocaudamer strategy. The recombinant integration plasmids pDG-nDPE (n = 1, 2, 3), which contained one, two, or three consecutive P43-DPEase tandem repeats, were integrated into the genome of B. subtilis. Then, the antibiotic resistance gene was deleted by the Cre/lox system, and the food-grade recombinant B. subtilis 1A751-nDPE (n = 1, 2, 3) with integrated tandem repeats of the P43-DPEase expression cassette were generated. The specific activity of the B. subtilis 1A751-3DPE was the highest among the recombinant strains and was ∼2.2-fold that of the 1A751-1DPE strain. Under the optimal conditions, 8 g/L of freeze-dried enzyme powder could convert 20% d-fructose (300 g/L) into d-allulose after 1 h.

Improved operational stability of d-psicose 3-epimerase by a novel protein engineering strategy, and d-psicose production from fruit and vegetable residues.

The aim of the present work was to improve stability of d-psicose 3-epimerase and biotransformation of fruit and vegetable residues for d-psicose production. The study established that N-terminal fusion of a yeast homolog of SUMO protein - Smt3 - can confer elevated optimal temperature and improved operational stability to d-psicose 3-epimerase. The Smt3-d-psicose 3-epimerase conjugate system exhibited relatively better catalytic efficiency, and improved productivity in terms of space-time yields of about 8.5kgL(-1)day(-1). It could serve as a promising catalytic tool for the pilot scale production of the functional sugar, d-psicose. Furthermore, a novel approach for economical production of d-psicose was developed by enzymatic and microbial bioprocessing of fruit and vegetable residues, aimed at epimerization of in situd-fructose to d-psicose. The bioprocessing led to achievement of d-psicose production to the extent of 25-35% conversion (w/w) of d-fructose contained in the sample.

A novel D-mandelate dehydrogenase used in three-enzyme cascade reaction for highly efficient synthesis of non-natural chiral amino acids.

A novel NAD(+)-dependent D-mandelate dehydrogenase was identified from Lactobacillus brevis (LbDMDH). After purified to homogeneity, the optimum pH and temperature for oxidation of D-mandelate were pH 10.0 and 40 °C, and the Km and kcat were 1.1 mM and 355 s(-1) respectively. Employing the LbDMDH together with a mandelate racemase from Pseudomonas putida and a leucine dehydrogenase (EsLeuDH) from Exiguobacterium sibiricum, we established a three-step one-pot domino reaction system for preparing chiral L-phenylglycine from racemic mandelic acid with internal cofactor recycling. Under the optimum conditions, 30.4 g rac-mandelic acid (0.2 M) at 1L scale had been converted into chiral L-phenylglycine, with 96.4% conversion, 86.5% isolation yield, >99% eep and 50.4 gL(-1)d(-1) space-time yield.

A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for D-psicose production: cloning, expression, purification, and characterization.

D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²âº. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using D-psicose as the substrate, the apparent K(m), k(cat), and catalytic efficiency (k(cat)/K(m)) were 27.4 mM, 49 s⁻¹, and 1.78 s⁻¹ mM⁻¹, respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production.

Characterization of a D-psicose-producing enzyme, D-psicose 3-epimerase, from Clostridium sp.

The gene coding for D-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co(2+) as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was D-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for D-psicose were 227 mM, 32,185 min(-1), and 141 min(-1 )mM(-1), respectively. At pH 8.0 and 55 °C, 120 g D-psicose l(-1) was produced from 500 g D-fructose l(-1) after 5 h.

Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine.

An isolate of a Pseudomonas sp. uses the L-NCC (N-carbamoyl-L-cysteine) pathway to convert DL-2-amino-Δ(2)-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine. Genes encoding ATC racemase (AtcA), L-ATC hydrolase (AtcB) and L-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(+). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of L-cysteine from DL-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for L-cysteine production from DL-ATC via the L-NCC pathway, enabling the complete L-NCC pathway to be elucidated.

Characterization of DcsC, a PLP-independent racemase involved in the biosynthesis of D-cycloserine.

The biosynthetic gene cluster responsible for the generation of the antibiotic D-cycloserine (DCS) has recently been disclosed. One of the putative enzymes described was DcsC, which showed a high degree of homology to diaminopimelate epimerase (DapF). Based on this homology, the activity of DcsC was presumed to be the racemization of O-ureido-L-serine, a proposed intermediate in DCS biosynthesis. Here we describe the cloning, overexpression and characterization of this enzyme. Using synthetic standards we show that DcsC is a racemase that operates on both O-ureido-L- and D-serine, and that it employs a two-base mechanism, with a thiolate-thiol pair in the active site. The activity of this enzyme was shown to be optimal at pH ~ 7.8, with a similar k(cat)/K(M) ratio in both the L→D direction and D→L direction. Activity was abolished with thiol-inactivating reagents such as iodoacetamide and Hg(2+) ions. Further evidence for a thiolate in the active site was obtained through the use of an epoxide-containing substrate analogue (6), which became covalently attached to the enzyme.

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785.

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of ß-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.

Characterization of a recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus and its application in the production of mannose from glucose.

A putative N-acyl-D-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min(-1) mg(-1) for D-glucose with a 47-kDa monomer. The epimerization activity for D-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as D-glucose, D-xylose, L-altrose, L-idose, and L-arabinose, to their C2 epimers, such as D-mannose, D-lyxose, L-allose, L-gulose, and L-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l(-1) and fructose at 47.5 g l(-1) were produced from 500 g l(-1) glucose at pH 7.5 and 75°C over 3 h by the enzyme.

Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10.

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.

Immunohistochemical application of D2-40 as basal cell marker in evaluating atypical small acinar proliferation of initial routine prostatic needle biopsy materials.

D2-40 has been recently discovered as a lymphatic endothelial cell marker, and some investigators have found that D2-40 is also expressed in myoepithelial cells of salivary gland or breast. In this study, we evaluated D2-40 expression of basal cells and applied D2-40 immunohistochemistry in the combination of P504S, cytokeratin 5, and p63 for ten lesions with atypical small acinar proliferation (ASAP) in initial prostatic needle biopsy. As a result, D2-40 was expressed in basal cells, lymphatic endothelial cells, and some stromal fibroblasts of normal prostatic tissue. Among ten ASAP lesions, the final diagnosis of seven lesions was resolved by combination immunohistochemistry. D2-40 was comparable to cytokeratin 5 and p63 as a basal cell marker, and there were no lesions that failed to provide an accurate final diagnosis using only D2-40 immunohistochemistry without cytokeratin 5 or p63. However, we found some D2-40-positive stromal fibroblasts or D2-40-positive lumen-collapsed lymphatic vessels neighboring atypical glands. Pathologists should pay attention to avoid recognizing these cells as basal cells. In conclusion, the combination of immunohistochemistry of P504S, cytokeratin 5, p63, and D2-40 may contribute to the accurate diagnosis of ASAP in the initial prostatic needle biopsy.

Purification of recombinant mandelate racemase: improved catalytic activity.

Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate and has been studied extensively as a model for understanding how enzymes catalyze the deprotonation of carbon acid substrates with relatively high pK(a) values. Purification of recombinant MR as a fusion protein with an N-terminal hexahistidine tag using immobilized-nickel ion affinity chromatography and elution with a linear gradient of EDTA revealed three enzyme species (mrI, mrII, and mrIII). While mrIII was catalytically inactive, both mrI and mrII catalyzed the racemization of (S)-mandelate with turnover numbers (k(cat)) of 190+/-22 and 940+/-24s(-1), respectively. Circular dichroism analysis suggested that mrIII was a partially unfolded or misfolded form of the enzyme. Replacement of the N-terminal hexahistidine tag by a StrepII-tag appeared to ameliorate the folding problem yielding a single enzyme species with a turnover number of 1124+/-43s(-1). The MR fusion protein bearing an N-terminal StrepII-tag and a C-terminal decahistidine tag also exhibited reduced turnover (k(cat)=472+/-37s(-1)). These results highlight a potential problem that may be encountered when producing fusion enzymes bearing a polyhistidine tag: soluble, active enzyme may be obtained but care must be taken to ensure that it is free of minor misfolded forms that can alter the apparent activity of the enzyme.

Recombinant human serine racemase: enzymologic characterization and comparison with its mouse ortholog.

D-serine plays a key role in glutamatergic neurotransmission in mammalian brain as a co-agonist of N-methyl-D-aspartate receptors. The enzyme responsible for D-serine biosynthesis, serine racemase (SR), is therefore a promising target for treatment of neuropathologies related to glutamate receptor excitotoxicity, such as stroke or Alzheimer's disease. Much of the experimental work to date has been performed on mouse serine racemase, which shares a high level of sequence identity with its human ortholog. In this work, we report the synthesis of a human SR gene variant optimized for heterologous expression in Escherichia coli and describe the expression and purification of active recombinant human SR. This strategy may be of general interest to researchers wishing to express mammalian proteins in a bacterial system. Furthermore, we conduct a thorough analysis of the kinetics and inhibitor-sensitivity of the recombinant enzyme, and we provide the first direct comparison of human and mouse SR based on our kinetic data. The orthologs behave similarly overall and exhibit identical inhibition profiles, validating the use of mouse models in SR research.

Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens.

Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).

Enzymatic properties of cellobiose 2-epimerase from Ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme.

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.