Purification and characterization of a recombinant laccase-like multi-copper oxidase from Paenibacillus glucanolyticus SLM1.

AIMS: The aim of this study was to evaluate the activity of a novel bacterial laccase-like multi-copper oxidase (LMCO) from Paenibacillus glucanolyticus SLM1: a bacterium isolated from pulp and paper waste. METHODS AND RESULTS: A new bacterial LMCO gene (CuOx) from P. glucanolyticus SLM1 was identified and cloned into pET22b. The protein it encodes was recombinantly expressed in Escherichia coli. The recombinant P. glucanolyticus LMCO had a molecular weight of approximately 90 kDa and demonstrated oxidation of the LMCO substrates 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), catechol, and 2,6-Dimethoxyphenol (2,6-DMP), with the oxidation of ABTS occurring to the greatest extent (776 U mg-1 under optimal conditions of pH 7 and 40°C). Furthermore, recombinant P. glucanolyticus CuOx retained activity against ABTS in the presence of 1 mol l-1 NaCl, 50% dimethyl sulfoxide and 5% Tween-80 and can decolorize several types of dyes. CONCLUSIONS: This enzyme has a neutral pH optimum, is capable of decolorizing dyes, and is active in the presence salt, detergents and surfactant. The characteristics of this enzyme suggest that it could be used for a variety of industrial applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This work characterizes a unique bacterial LMCO with activity higher than that of previously characterized fungal or bacterial LMCOs. This enzyme may have utility for industrial bleaching, treatment of dye effluent, and lignin removal.

Prolidase function in proline metabolism and its medical and biotechnological applications.

Prolidase is a multifunctional enzyme that possesses the unique ability to degrade imidodipeptides in which a proline or hydroxyproline residue is located at the C-terminal end. Prolidases have been isolated from archaea and bacteria, where they are thought to participate in proline recycling. In mammalian species, prolidases are found in the cytoplasm and function primarily to liberate proline in the final stage of protein catabolism, particularly during the biosynthesis and degradation of collagen. Collagen comprises nearly one-third of the total protein in the body, and it is essential in maintaining tissue structure and integrity. Prolidase deficiency (PD), a rare autosomal recessive disorder in which mutations in the PEPD gene affect prolidase functionality, tends to have serious and sometimes life-threatening clinical symptoms. Recombinant prolidases have many applications and have been investigated not only as a possible treatment for PD, but also as a part of anti-cancer strategies, a component of biodecontamination cocktails and in the dairy industry. This review will serve to discuss the many in vivo functions of procaryotic and eucaryotic prolidases, as well as the most recent advances in therapeutic and biotechnological application of prolidases.

Effects of antibiotics and oil on microbial profiles and fermentation in mixed cultures of ruminal microorganisms.

Ionophores and supplemental fat are fed to lactating cows to improve feed efficiency. Their effect on rumen fermentation is similar, but less is known about their impact on rumen microbes. The objective of this study was to determine the effects of monensin (M), bacitracin (B), and soybean oil (O) on microbial populations. Mixed cultures of rumen microbes were incubated in 5 dual-flow continuous fermentors and fed 13.8 g of alfalfa hay pellets daily (DM basis) for 16 d. All fermentors were allowed to stabilize for 4 d. From d 5 to 10, two fermentors received O (5% of diet DM), one fermentor received M (22 mg/kg), and one received B (22 mg/kg). From d 11 to 16, the 2 fermentors receiving O also received either M (OM) or B (OB) and O was included in the fermentors receiving M (MO) and B (BO). One fermentor served as the control and received 100% alfalfa pellets throughout the experiment. Each run was replicated 3 times. Samples were taken at 2 h after the morning feeding on d 4, 10, and 16 and were analyzed for bacterial populations using terminal restriction fragment length polymorphism. Volatile fatty acid concentration, methane production, and pH in the control cultures were not affected by time and remained similar during the entire experiment. The M and O treatments reduced molar concentration of acetate, increased concentration of propionate, and decreased methane production. Bacitracin did not alter acetate or propionate concentration, but reduced methane production. All 3 treatments (M, B, and O) altered the fragment patterns of microbial profiles. In contrast, treatments MO, OM, BO, and OB had little effect on culture fermentation despite differences in the patterns of microbial fragments. The terminal restriction fragment length polymorphism data suggest that microbial adaptation to the in vitro system in the control fermentor occurred within 4 d.

Molybdate transport.

In both bacteria and archaea, molybdate is transported by an ABC-type transporter comprising three proteins, ModA (periplasmic binding protein), ModB (membrane protein) and ModC, the ATPase. The modABC operon expression is controlled by ModE-Mo. In the absence of the high-affinity molybdate transporter, molybdate is also transported by another ABC transporter which transports sulfate/thiosulfate as well as by a nonspecific anion transporter. Comparative analysis of the molybdate transport proteins in various bacteria and archaea is the focus of this review.

Characterization of an aminoacylase from the hyperthermophilic archaeon Pyrococcus furiosus.

Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-L-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 +/- 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl(2) (and to 74% by the addition of CoCl(2)). After reconstitution with ZnCl(2), the enzyme contained 2.85 +/- 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolar N-acylated L amino acids (Met, Ala, Val, and Leu), as well as N-formyl-L-methionine. The high K(m) values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity with N-acetyl-L-methionine as the substrate occurred at pH 6.5 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in the P. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7 lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn(2+) ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of the P. furiosus aminoacylase (> or = 50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.

Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus.

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

Molybdate transport and regulation in bacteria.

Molybdate is transported in bacteria by a high-affinity transport system composed of a periplasmic binding protein, an integral membrane protein, and an energizer protein. These three proteins are coded by modA, modB, and modC genes, respectively. The ModA, ModB, and ModC proteins from various organisms (Escherichia coli, Haemophilus influenzae, Azotobacter vinelandii, and Rhodobacter capsulatus) are very similar. The lowest Km value reported for molybdate in the molybdate transport process is approximately 50 nM. In a mod mutant, molybdate is transported by the sulfate transport system or by a nonspecific anion transporter. Molybdate transport is tightly coupled to utilization in E. coli and Klebsiella pneumoniae, while other dinitrogen-fixing organisms appear to have a molybdenum storage protein. In all organisms studied so far, molybdate transport genes are regulated by a repressor protein, ModE. The ModE-molybdate complex binds to the sequences TAYAT (Y = T or C) in the operator/ promoter region in E. coli and prevents transcription of the modABCD operon. The ModE-molybdate complex binds to DNA as a homodimer in E. coli and possibly in other organisms as well. In R. capsulatus, however, two ModE homologues (MopAB proteins) are required for repression.

Repression of the Escherichia coli modABCD (molybdate transport) operon by ModE.

The modABC gene products constitute the molybdate-specific transport system in Escherichia coli. Another operon coding for two proteins which diverges from the modABCD operon has been identified. The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa), and the second genes codes for a 490-amino-acid protein. ModF (54 kDa). The role of ModF has not yet been determined; however, mutations in modE depressed modABCD transcription even in the presence of molybdate, suggesting that ModE is a repressor. ModE, in the presence of 1 mM molybdate, repressed the production of plasmid-encoded ModA and ModB' proteins in an in vitro transcription-translation system. DNA mobility shift experiments confirmed that ModE binds to an oligonucleotide derived from the operator region of the modABCD operon. Further experimentation indicated that ModE binding to target DNA minimally requires an 8-bp inverted-repeat sequence, TAAC GITA. A highly conserved amino acid sequence, TSARNOXXG (amino acids 125 to 133), was identified in ModE and homologs from Azotobacter vinelandii, Haemophilus influenzae, Rhodobacter capsulatus, and Clostridium pasterianum. Mutants with mutations in either T or G of this amino acid sequence were isolated as "superrepressor" mutants. These mutant proteins repressed modABCD transcription even in the absence of molybdate, which implies that this stretch of amino acids is essential for the binding of molybdate by the ModE protein. These results show that molybdate transport in E. coli is regulated by ModE, which acts as a repressor when bound to molybdate.