Keratinolytic abilities of Micrococcus luteus from poultry waste

Keratinolytic microorganisms have become the subject of scientific interest due to their ability to biosynthesize specific keratinases and their prospective application in keratinic waste management. Among several bacterial classes, actinobacteria remain one of the most important sources of keratin-degrading strains, however members of the Micrococcaceae family are rarely scrutinized in regard to their applicatory keratinolytic potential. The tested Micrococcus sp. B1pz isolate from poultry feather waste was identified as M. luteus. The strain, grown in the medium with 1–2% chicken feathers and a yeast extract supplement, produced keratinases of 32 KU and lower level of proteases, 6 PU. It was capable to effectively decompose feathers or “soft” keratin of stratum corneum, in contrast to other “hard” hair-type keratins. The produced keratinolytic enzymes were mainly a combination of alkaline serine or thiol proteases, active at the optimum pH 9.4, 55 °C. Four main protease fractions of 62, 185, 139 and 229 kDa were identified in the crude culture fluid. The research on the auxiliary role of reducing factors revealed that reducing sulfur compounds could be applied in keratinolysis enhancement during enzymatic digestion of keratin, rather than in culture conditions. The presented M. luteus isolate exhibits a significant keratinolytic potential, which determines its feasible applicatory capacity towards biodegradation of poultry by-products or formulation of keratin-based feed components.

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.

Topical liposomal DNA-repair enzymes in polymorphic light eruption.

Polymorphic light eruption (PLE) is a very frequent photodermatosis in Europe whose pathogenesis may involve resistance to UV-induced immune suppression and simultaneous immune reactions against skin photoneoantigens. We performed a randomized, double-blind, placebo-controlled intra-individual half-body trial to investigate the protective effect of an after-sun (AS) lotion containing DNA-repair enzymes (photolyase from Anacystis nidulans and Micrococcus luteus extract with endonuclease activity). Fourteen PLE patients were exposed to suberythemal doses of solar-simulated UV radiation on 4 consecutive days at 4 symmetrically located PLE-prone test fields per patient. The test fields were treated with (i) active AS lotion or (ii) a placebo lotion immediately after each UV exposure, or (iii) an SPF30 sunscreen before UV exposure or left untreated. All test fields were exposed to photoactivating blue light 1 h after each UV exposure. As shown by a newly established specific PLE test score (AA + SI + 0.4P [range, 0-12], where AA is affected area score [range, 0-4], SI is skin infiltration score [range, 0-4], and P is pruritus score on a visual analogue scale [range, 0-10]), PLE symptoms were significantly fewer on test sites treated with active AS lotion than on untreated (P = 0.00049) or placebo-treated test sites (P = 0.024). At 144 h after first UV exposure (the time point of maximal PLE symptoms), the mean test scores for untreated, active AS lotion-treated, and placebo-treated test fields were 4.39, 1.73 (61% reduction; 95% confidence interval (CI), 36% to 85%), and 3.20 (27% reduction; 95% CI, 3% to 51%), respectively. Pretreatment with SPF30 sunscreen completely prevented PLE symptoms in all patients. The present results indicate that DNA damage may trigger PLE and that the application of topical liposomes containing DNA repair enzymes to increase DNA repair may effectively prevent PLE.

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation.

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.

Crystal structure of salt-tolerant glutaminase from Micrococcus luteus K-3 in the presence and absence of its product L-glutamate and its activator Tris.

Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme. Our previous study determined the structure of its major 42-kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product L-glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a 'lid' part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for L-glutamate (G), 8 aa for Tris (T) and 6 aa for both L-glutamate and Tris (TG). L-glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate-binding site between Mglu and salt-labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate-binding pocket of Mglu, which is highly specific for L-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than L-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of L-asparagine with proper interactions.

Orientation-selective incorporation of transmembrane F0F1 ATP synthase complex from micrococcus luteus in polymer-supported membranes.

We report the vectorial incorporation of a highly asymmetric F0F1 ATP synthase complex from Micrococcus luteus into polymer-supported membranes. Dynamic light scattering and cryo electron microscopy confirm that the use of weak surfactants (bile acid) allows for the non-disruptive protein incorporation into lipid vesicles. Spreading of vesicles with ATP synthase onto a cellulose support results in a homogeneous distribution of proteins, in contrast to a patchy image observed on bare glass slides. The orientation of ATP synthase can be identified using an antibody to the ATP binding site as well as from topographic profiles of the surface. The method to "align" transmembrane proteins in supported membranes would open a possibility to quantify protein functions in biomimetic model systems.

Partial purification of mannosylphosphorylundecaprenol synthase from Micrococcus luteus: a useful enzyme for the biosynthesis of a variety of mannosylphosphorylpolyisoprenol products.

Membrane fractions from Micrococcus luteus catalyze the transfer of mannose from GDP-mannose to mono- and dimannosyldiacylglycerol, mannosylphosphorylundecaprenol (Man-P-Undec), and a membrane-associated lipomannan. This chapter describes the detergent solubilization, partial purification, and properties of Man-P-Undec synthase. The mobility of the mannosyltransferase activity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is a polypeptide with a molecular weight of approx 30.7 kDa. Utilizing the broad specificity of the bacterial mannosyltransferase provides a useful approach for the enzymatic synthesis of a wide variety of Man-P-polyisoprenol products.

UV-specific DNA repair recombinant fusion enzyme: a new stable pharmacologically active principle suitable for photoprotection.

BACKGROUND: UV radiation can produce mutations in skin cells and correlates strongly with the onset of actinic keratoses and basal and squamous cell carcinomas. Xeroderma pigmentosum (XP) is a heritable disease characterized by an extreme sensitivity of skin to UV radiation. Recently, studies in cultured cells as well as in XP patients have demonstrated that the recombinant T4 endonuclease V UV-specific endonuclease could enhance repair of UV-induced photoproducts. OBJECTIVE: We aimed to obtain a stable UV-specific DNA recombinant endonuclease, pharmacologically active in mammalian cells so as to be used in treatment and prophylaxis of sun damage. METHODS: The UV-specific DNA endonuclease gene obtained from Micrococcus luteus, was fused to a leader peptide and expressed (alphaUveA), refolded and purified. A construction under the control of an eukaryotic promoter was used to transfect XP fibroblasts deficient in DNA damage repair. Transformed cells were UV irradiated and cell survival was assessed. RESULTS: alphaUveA was obtained as a highly active UV-specific repair enzyme stable for at least 2 years. XP fibroblasts transfected with alphaUveA gene increased the resistance to UV radiation and, in consequence, cell survival. CONCLUSION: alphaUveA is stable and pharmacologically active in human cells. The topical administration of this long-term stable new active principle could help diminish the risks of skin cancer after sun exposure.

Active site analysis of P450 enzymes: comparative magnetic circular dichroism spectroscopy.

Recent structural studies indicate that the substrate- and O2-binding distal pocket of the P450 enzymes are not identical. Thus, P450terp (CYP108) from the alpha-terpineol-metabolizing Pseudomonad differs from P450cam (CYP-101) (C. A. Hasemann et al., J. Mol. Biol. 236, 1169, 1994). In contrast, the distal pockets of P450terp and P450BMP (CYP102 heme domain; Bacillus megaterium) are more closely similar, including novel hydrogen-bonding interactions between the distal H2O ligand and the I helix (C. A. Hasemann et al., Structure, 3, 41-62, 1995). To evaluate the significance of these differences, we have compared solution magnetic circular dichroism (MCD) spectra of P450terp with spectra of other P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase. Spectra of native P450terp are more similar to those of P450BMP and those of mammalian P450LM-2 than to those of P450cam. Upon substrate-binding, the MCD spectra of ferric P450terp and all other thiolate-ligated heme systems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or other 5-coordinate ferric heme systems. This intense negative MCD feature thus appears diagnostic for cysteinate-linked ferric hemes. In the case of ferrous P450s, the intensity of the Soret-region MCD trough varies between substrate-bound and substrate-free enzymes (despite the fact that the substrate is NOT in direct contact with the heme moiety). A novel finding of particular interest is the clear spectral shifts of the Soret MCD band between the substrate-bound and substrate-free forms of ferrous-CO-P450terp. No such observation has been made previously. Furthermore, the band positions for BOTH types of P450terp are red-shifted from known bands of ferrous-CO-P50cam. These data thus indicate a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on CO-binding rates and equilibrium constants. Comparative analysis of the spectral properties of P450terp with MCD spectra of other P450 enzymes, as well as with chloroperoxidase and NO synthase, demonstrates both the expected similarities and the significant differences that reflect active-site structural features. The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous-CO-P450. Furthermore, testable structural predictions for P450-BM-1 and for the novel NO synthase enzyme (neither of which has been crystallized to date) are made herein. This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes.

UV endonuclease of Micrococcus luteus, a cyclobutane pyrimidine dimer-DNA glycosylase/abasic lyase: cloning and characterization of the gene.

The gene of Micrococcus luteus UV endonuclease (cyclobutane pyrimidine dimer-DNA glycosylase/ abasic lyase) was cloned and characterized. The cloned gene, whose product had a predicted molecular mass of 17,120 Da, was found to be capable of complementing the Escherichia coli uvrA6 mutation in vivo with respect to resistance to acetonemediated molecular photosensitization, a treatment producing exclusively cyclobutane pyrimidine dimers in DNA. It also generated a nicking activity specific for photosensitization-treated DNA by in vitro transcription/translation. When expressed in E. coli cells, the gene produced a protein structurally identical with UV endonuclease and possessing an activity consistent with cyclobutane pyrimidine dimer-DNA glycosylase/abasic lyase with respect to the effect of inhibitors and the site of the DNA backbone scission. Furthermore, the UV endonuclease-deficient mutant DB7 was shown to regain the enzyme through transformation with the cloned gene. The deduced amino acid sequence of the gene product was at best 27% identical with that of endonuclease V of phage T4, an enzyme strikingly similar to UV endonuclease in molecular and catalytic properties. Despite this marginal overall similarity in amino acid sequence, four of the seven amino acid residues reported to be functionally important in the T4 enzyme were found to be conserved in the M. luteus enzyme. We propose that the gene be called uveA.

Photoaffinity labeling and photoaffinity crosslinking of enzymes.

Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme.

Photoaffinity labeling and photoaffinity crosslinking of enzymes

Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme

Reconstitution of bacteriorhodopsin and ATP synthase from Micrococcus luteus into liposomes of the purified main tetraether lipid from Thermoplasma acidophilum: proton conductance and light-driven ATP synthesis.

The archaebacterium Thermoplasma acidophilum is cultivated at 59 degrees C in a medium containing sulfuric acid of pH 2. The purified bipolar membrane spanning main phospholipid (MPL) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a French pressure cell detergent dialysis or sonication. Despite a potassium diffusion potential of 186 mV very low ionic permeability of sonicated MPL liposomes was measured using the potassium binding fluorescent indicator benzofuran isophthalate PBF1, which measures net K+ uptake. The latter also remained very low, in the presence of the K(+) ionophore valinomycin and palmitic acid. Addition of valinomycin and the potent uncoupler carbonylcyanid-p-trifluormehoxyphenyl-hydrazone (FCCP), led to a stimulation in potassium uptake. The rate of proton flux can be calculated from the net K(+) uptake. Under these conditions MPL liposomes are 1-2 orders of magnitude less permeable than egg yolk lecithin vesicles. The difference in proton permeability becomes even more pronounced with increasing temperature, examined using the fluorescent pH indicator pyranine. Purified bacteriorhodopsin from Halobacterium halobium was reconstituted into MPL liposomes in order to study the light-driven proton uptake in 150 mM KCl following addition of valinomycin, gramicidin, FCCP and Triton X-100. The light-driven proton transport into the liposomes was increased 30-fold by addition of valinomycin decreased by gramicidin and FCCP, and abolished by Triton X-100. Co-reconstituted MPL proteoliposomes containing bacteriorhodopsin and ATP synthase from Micrococcus luteus were capable of light-driven ATP synthesis demonstrating the functional coupling of proton transport and nucleotide generation in liposomal MPL membranes.

Enzymatic synthesis of polymers containing nicotinamide mononucleotide.

Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

Purification and characterization of the inhibitory subunit (delta) of the ATP-synthase from Micrococcus luteus.

Subunit delta was isolated from the ATP-synthase from Micrococcus luteus strain (ATCC 4698). delta, in the case of M. luteus F0F1-ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E. coli and chloroplast ATP-synthase. After treatment with 1.5 M LiCl the ATP-synthase dissociated, and subsequently subunit delta (27 kDa) was purified by hydrophobic interaction chromatography. Inhibition of ATP-synthase lacking delta by addition of delta showed non-competitive kinetics with a Ki of approximately 5.9 nM. Subunit epsilon from chloroplast F1, which corresponds functionally to the M. luteus F0F1-delta, and chloroplast delta were tested for ATPase inhibitory activity by addition to the partially delta-depleted ATP-synthase from M. luteus. CF1-epsilon inhibited M. luteus ATP-synthase up to 80%, whereas CF1-delta did not show any influence.

ATP synthesis and hydrolysis of the ATP-synthase from Micrococcus luteus regulated by an inhibitor subunit and membrane energization.

After incubation for 70 min in Tris-HCl (pH 8.0), the rate of ATP hydrolysis of free and reconstituted ATP-synthase from Micrococcus luteus multiplied about three times. The apparent increase in activity is due to the reversible dissociation of the delta-subunit. Results of experiments on the temperature dependence of the ATP hydrolysis rate of substrate saturated ATP-synthase exhibited a discontinuity in the Arrhenius plot at 32 +/- 0.5 degrees C for the delta-subunit associated enzyme. Below 32 +/- 0.5 degrees C the activation energy, Ea, was 231.5 +/- 5 kJ mol-1, while above this temperature-level it decreased to 76.4 +/- 3 kJ mol-1. ATP synthesis and hydrolysis of the ATP-synthase, co-reconstituted with monomeric bacteriorhodopsin (Halobacterium halobium), showed a lag of 50 s upon the illumination with green light (505-575 nm). This retardation and the activity depended on the ATP-synthase concentration, being typical of the dissociation of an inhibitor protein. The N-terminal protein sequences of the delta- and epsilon-subunit of the ATP-synthase were identified by automated Edman degradation. Alignment of the amino acid sequence and secondary structure calculations for the delta-subunit did not reveal homology to other known ATP-synthase delta-subunits, but significant equivalence to the epsilon-subunit of E. coli. Sequence analysis of the epsilon-subunit from M. luteus showed homology to equivalent regions in delta-subunits and Oligomycin Sensitivity Conferring Protein (OSCP) of other organisms.

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation.

Quantitation of UV-induced DNA damages in nanogram quantities of non-radioactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.

Purification of solanesyl-diphosphate synthase from Micrococcus luteus. A new class of prenyltransferase.

The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography. By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa. In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate. HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner. Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis.