Construction and characterization of the GFAT gene as a novel selection marker in Aspergillus nidulans.

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.

Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2.

His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology.

The bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreductase from Thermococcus guaymasensis.

The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic ß -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding ß -keto acids.

A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon Hyperthermus butylicus.

Hyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in H. butylicus to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell-free extract of H. butylicus, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H. butylicus. HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as k c at/K m, with k cat and apparent K m values of 8.00 ± 0.22 s-1 and 0.59 ± 0.07 mM, respectively. The apparent K m values for other substrates, including ethanol, 1-propanol, 2-propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1-butanol in H. butylicus. The putative conserved motif sites for NAD(P)+ and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.

Molecular Characterization of the Iron-Containing Alcohol Dehydrogenase from the Extremely Thermophilic Bacterium Pseudothermotoga hypogea.

Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/ß-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs.

Thermostable and O2-Insensitive Pyruvate Decarboxylases from Thermoacidophilic Archaea Catalyzing the Production of Acetaldehyde.

Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen.

Characterization of Thermotoga neapolitana Alcohol Dehydrogenases in the Ethanol Fermentation Pathway.

Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.

Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis.

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP(+). Metal analyses revealed that this NADP(+)-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K(m) values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP(+), respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.

A novel bifunctional aldehyde/alcohol dehydrogenase catalyzing reduction of acetyl-CoA to ethanol at temperatures up to 95 °C.

Hyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80-85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Characterization of a thioredoxin-thioredoxin reductase system from the hyperthermophilic bacterium Thermotoga maritima.

A thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract of Thermotoga maritima. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purified T. maritima thioredoxin reductase could not use thioredoxin from Spirulina as an electron acceptor, it used thioredoxin that was purified from T. maritima by monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparent V(max) values of 1,111 +/- 35 micromol NADH oxidized min(-1)mg(-1) and 115 +/- 2.4 micromol NADPH oxidized min(-1)mg(-1), respectively. The apparent K(m) values were determined to be 89 +/- 1.1 microM, 73 +/- 1.6 microM, and 780 +/- 20 microM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95 degrees C, and more than 60% of the activity remained after incubation for 28 h at 80 degrees C. The purified T. maritima thioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities. T. maritima thioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5'-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.

Applications of Microbial ß-Mannanases.

Mannans are main components of hemicellulosic fraction of softwoods and they are present widely in plant tissues. ß-mannanases are the major mannan-degrading enzymes and are produced by different plants, animals, actinomycetes, fungi, and bacteria. These enzymes can function under conditions of wide range of pH and temperature. Applications of ß-mannanases have therefore, been found in different industries such as animal feed, food, biorefinery, textile, detergent, and paper and pulp. This review summarizes the most recent studies reported on potential applications of ß-mannanases and bioengineering of ß-mannanases to modify and optimize their key catalytic properties to cater to growing demands of commercial sectors.

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1.

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45+/-1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0+/-0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105+/-2 U mg(-1), which was very similar to that of the native enzyme (110+/-3 U mg(-1)). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K (m)-values for acetaldehyde and NADPH and higher K (m)-values for ethanol and NADP(+). It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.

Degradation of Perfluorooctane Sulfonamide by Acinetobacter Sp. M and Its Extracellular Enzymes.

The Acinetobacter sp. strain M isolated from a contaminated soil sample in Jiangsu Province of China was found to be able to degrade perfluorooctane sulfonamide (PFOSA) effectively. Fluoride anion (F- ) released from PFOSA degradation was detected by ion chromatography, and showed positive correlation to the growth curve of Acinetobacter sp. strain M. The PFOSA degradation efficiency of strain M was approximately 27 %, as assessed by GC analysis. It was shown that enzymes localized outside of cells of Acinetobacter sp. strain M catalyzed the degradation of PFOSA. This further indicates a possibly new (multi-step/pathway) mechanism for PFOSA degradation. It revealed that the extracellular enzyme of the Acinetobacter strain M preferentially cleaves carbon-carbon and carbon-fluorine bonds instead of destroying the carbon-sulfur bond. The growth condition for Acinetobacter sp. strain M was optimized at 30 °C and pH 7.0 in the presence of 2000 mg L-1 of PFOSA and 0.5 % (v/v) of Tween-20. The optimal PFOSA degradation time was found to be 12 h, with a degradation efficiency of 76 % by extracellular enzymes in strain M as determined by GC analysis. The result may provide potential applications for biodegradition of perfluoro organic compounds, such as derivatives of perfluorooctane (C8).

Metagenomic Sequencing of Wastewater from a South African Research Farm.

We sequenced wastewater effluent from the Agricultural Research Council-Animal Production in South Africa that conducts studies on livestock health and farm ecology. Thauera, Oscillibacter, and Pseudomonas were the most abundant genera within the community. Thirty-one different antibiotic resistance genes were identified, 10 of which are associated with tetracycline resistance.

Draft Genome Sequences of Two Novel Cellulolytic Streptomyces Strains Isolated from South African Rhizosphere Soil.

We report here the draft genome sequences of two novel strains of Streptomyces (NWU339 and NWU49) isolated from South African rhizosphere soils. Both strains were found to possess strong cellulolytic activity and contain numerous putative cellulase genes. Both genomes possess benzoate degradation pathways, while NWU49 contains the genomic potential for enediyne biosynthesis.

Characterization of the diethyl phthalate-degrading bacterium Sphingobium yanoikuyae SHJ.

A newly isolated bacterial strain SHJ was found to be capable of degrading diethyl phthalate (DEP) very efficiently. Its growth characteristics and 16S rDNA gene sequence were analyzed. Its whole genome was also sequenced. Strain SHJ was identified as Sphingobium yanoikuyae SHJ.

High Production of 2,3-butanediol by a Mutant Strain of the Newly Isolated Klebsiella pneumoniae SRP2 with Increased Tolerance Towards Glycerol.

Biodiesel, a renewable fuel produced by transesterification of animal fats and vegetable oils, generates about 10% (v/v) of crude glycerol as a core byproduct. The high volume of this non bio-degradable glycerol is becoming of a great environmental and economical concern due to its worldwide ever-growing surplus. Herein we report a high production of 2,3-butanediol (2,3-BD) from pure and biodiesel derived crude glycerol using a mutant K. pneumoniae SRM2 obtained from a newly isolated strain Klebsiella pneumoniae SRP2. The mutant strain SRM2 with standing high glycerol concentration (220 g L-1 of medium) could rapidly convert glycerol aerobically to 2,3-BD, a versatile product extensively used in chemical, pharmaceutical and fuel industries Our study revealed that an increased GDH activity led to a substantially enhanced production of 2,3-BD. The mutant strain exhibited 1.3-fold higher activity of GDH than that of parent strain (500.08 vs. 638.6 µmol min -1 mg -1 protein), yielding of 32.3 g L-1 and 77.5 g L-1 2,3-BD with glycerol in batch and fed-batch process respectively. However, in batch culture with crude glycerol, cell growth and glycerol consumption were expressively boosted, and 2,3-BD production was 27.7 g L-1 from 75.0 g/L crude glycerol. In this report, the optimal conditions for high production of 2,3-BD were defined in a completely aerobic process, and 0.59 g g-1 product yield of 2,3-BD was attained by the mutated strain K. pneumoniae SRM2, which is the highest amount obtained from batch biotransformation process of glycerol metabolism till today. These results indicated that our newly developed mutant can tolerate high concentration of glycerol, have a high glycerol utilization rate, and high product yield of 2,3-BD. It is demonstrated that the mutant strain K. pneumoniae SRM2 has an ability to produce fewer co-products at trace concentrations at higher glycerol concentrations, and could be a potential candidate for 2,3-DB production in an industrial bioconversion process.

Development and Use of a Novel Random Mutagenesis Method: In Situ Error-Prone PCR (is-epPCR).

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.

Ag-Initiated gem-Difluoromethylenation of the Nitrogen Center of Arenediazonium Salts to gem-Difluoromethylene Azo Compounds.

An efficient method for the synthesis of the thermally stable and pharmaceutically important gem-difluoromethylene azo compounds is developed. This protocol achieved gem-difluoromethylenation of the nitrogen center of arenediazonium salts through in situ generated benzo-1,3-diazolic difluoromethylene radical addition to arenediazonium salts under mild Ag-initiated conditions.

Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease.

HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ßßα-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra α-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.