The Response Regulator FimR Is Essential for Fimbrial Production of the Oral Anaerobe Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative oral anaerobe considered to be a major pathogen in adult periodontitis. One of the noted virulence factors of this bacterium is its unique fimbriae, which are composed of FimA (fimbrilin) as a major subunit. We have recently identified and isolated two essential genes, fimS and fimR, for fimbriation of P. gingivalis from transposon-mutagenesis studies. The genes encode two components of a His–Asp phosphorelay system, FimS as a sensor histidine kinase and FimR as a response regulator. Disruption of either gene causes fimbrial deficiency in this organism. In this study, the expression of FimR protein was detected in various P. gingivalis strains. In addition, a fragment containing fimR with a possible promoter was introduced into the fimR -disruption mutant, using a shuttle vector, pT-COW. The transconjugant recovered both FimR and FimA expression at levels comparable to the parentP. gingivalis ATCC 33277. Furthermore, characteristic fimbrial structures were clearly observed around the cell surface of both parent and transconjugant cells under electron microscopy. This is the first successful complementation experiment in P. gingivalis. These results show that the FimR protein is essential as a positive regulator in fimbriation of P. gingivalis.     

PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H₂O₂, whereas Dpr (an Fe²⁺-chelating protein)-dependent H₂O₂ protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H₂O₂-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl₂, S. oligofermentans increased survival after H₂O₂ stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn²⁺-mediated H₂O₂ resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn²⁺) unless it was supplemented with ≥2.5 μM MnCl₂ and caused 82 to 95% reduction of the cellular Mn²⁺ level, while mntABC overexpression increased cellular Mn²⁺ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn²⁺ transporter in S. oligofermentans. mntA mutation reduced the survival of H₂O₂-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H₂O₂-challenged survival 12-fold, indicating that MntABC-mediated Mn²⁺ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H₂O₂ pulsing upregulated mntABC, while H₂O₂-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H₂O₂ can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H₂O₂ stress defenses and may be distributed across all oral streptococci lacking catalase.     

Transcriptional Regulation,Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn²⁺ and Mn²⁺ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn²⁺ or Mn²⁺ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn²⁺ over Mn²⁺ specificity, the Zn²⁺ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn²⁺-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn²⁺-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn²⁺-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn²⁺ specificity.     

Hymenolepis diminuta: Partial characterization of the membrane-bound and solubilized alkaline phosphohydrolase activities of the isolated brush border plasma membrane

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate (p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg²⁺ (1 mM), although low concentrations of Mn²⁺, Ca²⁺, or Zn²⁺ will also partially satisfy this requirement; higher concentrations of Mg²⁺ and Mn²⁺, and other divalent cations (Cu²⁺, Fe²⁺, and Pb²⁺), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and K_m, but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F^?, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.     

The effect of Mn2+ on ethanol production from lactose using Kluyveromyces marxianus IMB3 immobilized in magnetically responsive matrices

The thermotolerant, ethanol producing yeast strain, K. marxianus IMB3 was immobilized in calcium alginate containing magnetically responsive Fe₃O₄ particles. In these studies the β-galactosidase derived from K. marxianus IMB3 was immobilized onto the Fe₃O₄ particles prior to inclusion into the alginate matrix. Ethanol production by the immobilized microorganism in the presence of Fe₃O₄ reached a maximum of 16?g/L on 40?g/L lactose whereas prior immobilization of the enzyme to the particles and inclusion into the alginate matrix increased ethanol production to a maximum concentration of 18 g/L. When Mn²⁺ was incorporated into fermentations containing the immobilized enzyme in the alginate matrix, ethanol production increased further to a maximum concentration of 20?g/L. In addition, the behaviour of the magnetically responsive biocatalyst containing the co-immobilized enzyme was examined in a batch-fed system in the presence and absence of Mn²⁺.     

Enzymatic characteristics of an ApaH-like phosphatase,PrpA, and a diadenosine tetraphosphate hydrolase,ApaH, from Myxococcus xanthus

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap₄A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap_nA and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap₄A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap₄A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap_nA and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap_nA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap_nA hydrolase, respectively.     

Altering the Divalent Metal Ion Preference of RNase E

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg²⁺ and Mn²⁺ will support significant rates of activity in vitro against natural RNAs, with Mn²⁺ being preferred. Both Mg²⁺ and Mn²⁺ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni²⁺ and Zn²⁺ permitted low levels of activity, while Ca²⁺, Co³⁺, Cu²⁺, and Fe²⁺ did not. A mutation to one of the residues known to chelate Mg²⁺, D346C, led to almost complete loss of activity dependent on Mg²⁺; however, the activity of the mutant enzyme was fully restored by the presence of Mn²⁺ with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO₄, implying that RNase E relies on Mg²⁺ exclusively in vivo.     

Influence of Manganese on Growth of a Sheathless Strain of Leptothrix discophora

Mn²⁺ exerted various effects on the growth of Leptothrix discophora strain SS-1 in batch cultures depending on the concentration added to the medium. Concentrations of 0.55 to 5.5 μM Mn²⁺, comparable to those in the environment from which strain SS-1 was isolated, decreased cell yield and prolonged stationary-phase survival, but did not affect growth rate. Elevated concentrations of 55 to 910 μM Mn²⁺ also decreased cell yield and prolonged survival, but growth rate was decreased as well. The addition of 1,820 μM Mn²⁺ caused a decline in cell numbers followed by an exponential rise after 80 h of incubation, indicating the development of a population of cells resistant to Mn²⁺ toxicity. When 360 μM Mn²⁺ or less was added to growth flasks, Mn²⁺ was oxidized to manganese oxide (MnO_x, where x is ~2), which appeared as brown particles in the medium. Quantification of Mn oxidation during growth of cultures to which 55 μM Mn²⁺ was added showed that nearly all of the Mn²⁺ was oxidized by the beginning of the stationary phase of growth (15 to 25 h). This result suggested that the decrease in cell yield observed at low and moderate concentrations of Mn²⁺ was related to the formation of MnO_x, which may have bound cationic nutrients essential to the growth of SS-1. The addition of excess Fe³⁺ to cultures containing 55 μM Mn²⁺ increased cell yield to levels near those found in cultures with no added Mn²⁺, indicating that iron deprivation by MnO_x was at least partly responsible for the decreased cell yield.     

Production and characterization of laccase from Pleurotus ferulae in submerged fermentation

Pleurotus ferulae is a mushroom typically found in arid steppe that is distributed widely in the Junggar Basin of Xinjiang, China. In this work, laccase production by P. ferulae JM30X was optimized in terms of medium composition and culture conditions. After optimization, the highest laccase activity obtained was 6,832.86 U/L. A single isozyme with a molecular weight of 66 kDa was observed by SDS-PAGE and native-PAGE. Optimum pH and temperature were 3.0 and 50–70 °C, respectively. The best laccase substrate was ABTS, for which the Michaelis-Menten constant (K _m) and catalytic efficiency (K _cat/K _m) value for P. ferulae laccase were 0.193 mM and 2.73?×?10⁶ (mM s)^?1, respectively. The activity of purified laccase was increased by more than four-fold by Cu²⁺, Mn²⁺ and Mg²⁺, while it was completely inhibited by Fe²⁺ and Fe³⁺. The production of laccase was influenced by the initial pH and K⁺ concentration, and the activity of purified laccase was enhanced by Cu²⁺, Mn²⁺ and Mg²⁺. This Pleurotus genus laccase from P. ferulae JM30X was analyzed by MS spectrum and the results are conducive to furthering our understanding of Pleurotus genus laccases.     

Manganese Supplementation Reduces High Glucose-induced Monocyte Adhesion to Endothelial Cells and Endothelial Dysfunction in Zucker Diabetic Fatty Rats

Endothelial dysfunction is a hallmark of increased vascular inflammation, dyslipidemia, and the development of atherosclerosis in diabetes. Previous studies have reported lower levels of Mn²⁺ in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. This study examines the hypothesis that Mn²⁺ supplementation can reduce the markers/risk factors of endothelial dysfunction in type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured with or without Mn²⁺ supplementation and then exposed to high glucose (HG, 25 mm) to mimic diabetic conditions. Mn²⁺ supplementation caused a reduction in monocyte adhesion to HUVECs treated with HG or MCP-1. Mn²⁺ also inhibited ROS levels, MCP-1 secretion, and ICAM-1 up-regulation in HUVECs treated with HG. Silencing studies using siRNA against MnSOD showed that similar results were observed in MnSOD knockdown HUVECs following Mn²⁺ supplementation, suggesting that the effect of manganese on monocyte adhesion to endothelial cells is mediated by ROS and ICAM-1, but not MnSOD. To validate the relevance of our findings in vivo, Zucker diabetic fatty rats were gavaged daily with water (placebo) or MnCl₂ (16 mg/kg of body weight) for 7 weeks. When compared with placebo, Mn²⁺-supplemented rats showed lower blood levels of ICAM-1 (17%, p < 0.04), cholesterol (25%, p < 0.05), and MCP-1 (28%, p = 0.25). These in vitro and in vivo studies demonstrate that Mn²⁺ supplementation can down-regulate ICAM-1 expression and ROS independently of MnSOD, leading to a decrease in monocyte adhesion to endothelial cells, and therefore can lower the risk of endothelial dysfunction in diabetes.     

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

Determination of the growth and solubilization capabilities of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> T1

The growth capability of Trichoderma harzianum Rifaii Tl was tested on Malt Extract and Czapeks Dox agar containing different concentrations of Cu²⁺, Zn²⁺, Mn²⁺, Fe²⁺ and Ca²⁺. The T. harzianum Tl isolate was observed to produce mycelia and spores in various mineral-containing media. It showed the lowest tolerance to Ca²⁺ and the highest tolerance to Fe²⁺. Solubilization capability of T. harzianum Tl for some insoluble minerals via acidification of medium has been tested on MnO₂, CuO, Fe₂O₃ and metallic Zn. T. harzianum Tl was able to solubilize MnO₂ and metallic Zn in a liquid medium.     

Magnetic resonance studies of the mitochondrial divalent cation carrier

Measurements of water proton spin relaxation enhancements (ε) can be used to discriminate high-affinity binding of Mn²⁺ or Gd³⁺ to biological membranes, from low-affinity binding. In rat liver mitochondria, ε_b values of approx. 11 are observed upon binding of Mn²⁺ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn²⁺ uptake by liver mitochondria results in the subsequent decay of ε¹.Comparison of ε¹ with the initial velocity of Mn²⁺ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 °C and 40 °C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn²⁺ to the divalent cation pump.Binding of Mn²⁺ to blowfly flight muscle also results in substantial ε¹, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in ε¹ due to uptake occurs after Mn²⁺ is bound.Lanthanide ions are also bound and transported by mitochondria. Addition of Gd³⁺ to pigeon heart or rat liver mitochondria results in ε_b ≈ 5–6, which decays with similar kinetics in both systems. The uptake velocity of Gd³⁺ in rat liver mitochondria is about $\text{1}\text{6}$ the rate with which Mn²⁺ is transported. Lanthanides also diminish ε¹ due to the addition of Mn²⁺, and greatly retard the Mn²⁺ uptake kinetics. The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses ε¹ upon addition of Mn²⁺ or Gd³⁺ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn²⁺, but does not affect ε¹ significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of “de-energization”.Measurements of ε¹ at several NMR frequencies indicate a correlation time (τ_b) for carrier-bound Mn²⁺ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 °C and 30 °C. The 13 Kcal/mole activation energy for τ_b suggests that the 11 ns time constant at room temperature represents the movement of the Mn^II-carrier complex. On the other hand, τ_b is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn²⁺ binds to a small arm of the carrier which moves independently of the main body of any protein.In addition to Mn(H₂O)₆²⁺, other complexes of Mn²⁺ may also be bound and transported by rat liver mitochondria. Only a small increase in ε¹ occurs upon addition of MnHPO₄, yet this species is accumulated by the mitochondria. Consequently, the carrier does not recognize divalent metal ions on the basis of charge.     

Differences in the cation sensitivity of adenylate cyclase from heart and skeletal muscle: modification by guanyl nucleotides and isoproterenol

Low concentrations of Mn²⁺ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg²⁺; at saturating concentrations the cyclase activities obtained with Mg²⁺ or Mn²⁺ were similar. In contrast, Mg²⁺ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn²⁺; at saturating concentrations of either metal ion the Mg²⁺-supported cyclase activities were 5- to 10-fold greater than Mn²⁺-supported activities. Further, compared to Mg²⁺, Mn²⁺ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg²⁺ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn²⁺ in heart as well as skeletal muscle appeared to be greater compared to those for Mg²⁺. The skeletal muscle cyclase displayed greater apparent affinity for MnATP^2? (app. K_m 0.10 mm) compared to MgATP^2? (app. K_m 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP^2? (app. K_m 0.07 mm) compared to MnATP^2? (app. K_m 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn²⁺ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg²⁺. With the heart enzyme the relatively greater potency of Mn²⁺ persisted following preactivation. Significant enhancement in the Mn²⁺-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me²⁺ (Mg²⁺ or Mn²⁺) without influencing the apparent K_m for MeATP^2? (MgATP^2? or MnATP^2?). Evidences were obtained to show that the poor effectiveness of Mn²⁺ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn²⁺ of adenosine-mediated inhibition of the cyclase, (b) Mn²⁺-induced lability of the cyclase, (c) indirect effects of Mn²⁺ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn²⁺ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

Characterization of a novel lipase and its specific foldase from Acinetobacter sp. XMZ-26

A novel operon containing a lipase gene (lip26) and its specific foldase gene (lif26) was discovered from Acinetobacter sp. XMZ-26 by creating and screening a gene library and then using genome walking. The amino acid sequence of Lip26 and Lif26 showed only 46.4% and 37.3% identity with the LipA and LipB (Lif) sequences from Acinetobacter sp. SY-01, respectively. The expressed recombinant Lip26 formed inactive inclusion bodies in Escherichia coli. However, the active Lip26 was refolded by the dilution refolding method with the assistance of purified recombinant Lif26, and the refolded Lip26 had a high specific activity. Lip26 hydrolyzed p-nitrophenyl (pNP) esters of fatty acids with C₂ to C₁₆ acyl chain lengths and had a substrate preference for pNP myristate. Maximal Lip26 activity was dependent on both the temperature (55 °C) and pH (9.0). In addition, Lip26 was capable of maintaining its activity in the presence of many detergents and organic solutions, and its activity was enhanced by the presence of Ca²⁺, Mn²⁺, and Ba²⁺. To directly obtain active Lip26, an E. coli strain was co-transformed with two expression plasmids containing the lip26 and lif26 genes. The co-expression of both proteins in vivo resulted in the expression of half of the recombinant Lip26 as a soluble protein with demonstrable lipase activity. A direct protein interaction between Lif26 and Lip26A was detected by both a pull-down assay and a yeast two-hybrid experiment.     

Substrate and metal ion binding to carbamate kinase: NMR and EPR studies

Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn²⁺ as the paramagnetic probe. The enzyme binds Mn²⁺ weakly (K_D = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn²⁺ was observed with K_D = 44 ± 4 μm in the presence of ADP and K_D = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn²⁺-nucleotide and binary enzyme-Mn²⁺ complexes. Mn²⁺ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?_b = 9.3 ± 0.4. When enzyme-Mn²⁺ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn²⁺-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?_t^ADP = 14 ± 1 and ?_t^ATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K₁ = 2.5 ± 0.5 μm and K₁ = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with ¹³C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn²⁺. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase.