Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.     

Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2

A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N′-diacetylchitobiose were 1.06 mM and 44.4 μmol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N′-diacetylchitobiose, indicating that ChiA is a chitobiosidase. Received: 29 October 1999 / Received revision: 16 March 2000 / Accepted: 24 March 2000     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

Purification and characterization of a novel enzyme, L-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62

L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3-hydroxyaspartate (K(m)=0.74 mM, V(max)=37.5 micromol min(-1) (mg protein)(-1)) but not for D-threo or D, L-erythro-3-hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5'-phosphate and divalent cations participate in the enzyme reaction. The NH(2)-terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity.     

Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2.

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.     

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.     

Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.

A cell-bound cyclodextrin-degrading enzyme with a relative molecular mass (M_r) of around 62 000 and an isoelectric point (pI) near 8.0 was isolated and purified to 94% homogeneity from Flavobacterium sp. The enzyme hydrolysed maltooligosaccharides and cyclodextrins to glucose, maltose, and maltotriose. Less glucose, but larger amounts of the line of maltooligosaccharides from maltose to (in case of cyclodextrins) the linearized substrates were found in short-term digests. Digestion of maltotriose yielded glucose, maltose, and some maltotetraose to maltohexaose, i.e. the enzyme catalysed both hydrolysis and transglycosylation. Starch was a poorer substrate, and was hydrolysed to mainly glucose and maltose, presumably by a kind of exo-attack. Pullulan was slightly digested, the products being glucose, panose/isopanose, and larger saccharides containing -1,6-glucosidic bonds. Since maltohexaose to maltooctaose were hydrolysed at higher rates than the cyclodextrins of corresponding lengths, the enzyme of Flavobacterium sp. was proposed to be classified as a decycling maltodextrinase. Correspondence to: H. Bender     

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase: immunochemical characterization of the lung enzyme from pregnant rabbits

A polyclonal antibody was produced in guinea pig against the lung NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.     

Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa.

A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.     

Purification and characterization of butyrylcholine-hydrolyzing enzyme from Pseudomonas polycolor.

A butyrylcholine-hydrolyzing enzyme (EC 3.1.1.-) fo Pseudomonas polycolor IFO 3918 was purified approximately 9270-fold with a recovery of 9.9% by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoretic and ultracentrifugational analyses. The molecular weight was determined as approximately 59000 by gel filtration. Isoelectric focusing electrophoresis revealed that the enzyme had an isoelectric point around pH 5.1. The enzyme catalyzed the hydrolysis of butyrylcholine with the miximum activity among various esters tested, and split benzoylcholine, propionylcholine and some aliphatic esters, but did not attact acetylcholine. The estimated value of Km at pH 7.5 and 25 degrees C was 7-10(-4) M for butyrylcholine. The enzyme was irreversibly inhibited by organophosphorus compounds and carbamates, such as diisopropylphosphofluoridate and eserine. The enzyme was inhibited by some compounds, such as atropine and quinidine. Auaternary ammonium salts showed an inhibitory effect on the enzyme resembling co-operative inhibition.     

Isolation, purification, and characterization of a new enzyme from Pseudomonas sp. M-27, carboxypeptidase G3.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M(r): 15,000). The enzyme hydrolyzed predominantly acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.     

Purification and characterization of a staphylolytic enzyme from Pseudomonas aeruginosa

A strain of Pseudomonas aeruginosa has been shown to produce an enzyme that lyses viable cells of Staphylococcus aureus. The maximal yield of the enzyme was obtained from shake flask cultures of P. aeruginosa which were grown for 18 to 22 hr at 37 C in Trypticase Soy Broth. A 333-fold purification of the enzyme was obtained by acetone precipitation of the culture liquor, followed by column chromatography on phosphonic acid cellulose and Bio-Gel P2. The staphylolytic enzyme exhibited maximal activity at 37 C in 0.01 m sodium phosphate (pH 8.5) and was stable at 37 C in the pH range of 7.5 to 9.5. The inhibition and stabilization of the enzyme by various organic and inorganic materials was investigated. Spheroplasts of S. aureus were formed by treating viable cells with the staphylolytic enzyme in 1 m sucrose or human serum.     

Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit

NAD+-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The Km, values for sorbitol and fructose were 96.4+/-8.60 and 4239+/-33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.     

Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.     

Purification and characterization of 2'aminobiphenyl-2,3-diol 1,2-dioxygenase from Pseudomonas sp. LD2

Carbazole is a nitrogen-containing heteroaromatic compound that occurs as a widespread and mutagenic environmental pollutant. The 2'aminobiphenyl-2,3-diol 1,2-dioxygenase involved in carbazole degradation was purified to near electrophoretic homogeneity from Pseudomonas sp. LD2 by a combination of ion-exchange chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. This purification was challenging due to the great instability of the enzyme under many standard conditions. The enzyme was also purified to electrophoretic homogeneity from recombinant Escherichia coli expressing the 2'aminobiphenyl-2,3-diol 1,2-dioxygenase-encoding gene cloned from Pseudomonas sp. LD2. The molecular mass of the native enzyme was determined by gel filtration to be 70 kDa. The subunit molecular masses were determined to be 25 and 8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase is an [alpha2beta2] heterotetramer. The optimal temperature and pH for the enzymatic production of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) from 2,3-dihydroxybiphenyl were determined to be 40 degrees C and 8.0, respectively. The maximum observed specific activity on 2,3-dihydroxybiphenyl was 48.1 mmol HOPDA min(-1) mg(-1). This indicated a maximum observed turnover rate of 360,000 molecules HOPDA enz(-1) s(-1). The K'm inhibition constant Ks and Vmax on 2,3 dihydroxybiphenyl were determined to be 5 microM, 37 microM, and 44 mmol min(-1) mg(-1), respectively. These results show that 2'aminobiphenyl-2,3-diol 1,2-dioxygenase is a meta-cleavage enzyme related to the 4,5-protocatechuate dioxygenase family, with comparable purification challenges posed by intrinsic enzyme instability.     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

Purification and characterization of a novel enzyme, N-carbamoylsarcosine amidohydrolase, from Pseudomonas putida 77

N-Carbamoylsarcosine amidohydrolase, a novel enzyme involved in the microbial degradation of creatinine in Pseudomonas putida 77, was purified 27-fold to homogeneity with a 63% overall recovery through simple purification procedures including successive ammonium sulfate fractionation, DEAE-cellulose chromatography, and crystallization. The relative molecular mass of the native enzyme estimated by the ultracentrifugal equilibrium method is 102,000 +/- 5000, and the subunit Mr is 27,000. The Km and Vm values for N-carbamoylsarcosine are 3.2 mM and 1.75 units/mg protein, respectively. Ammonia, carbon dioxide, and sarcosine were formed stoichiometrically from N-carbamoylsarcosine through the action of the purified enzyme preparation. N-Carbamoyl amino acids with a methyl group or hydrogen atom on the amino-N atom and possessing glycine, D-alanine, or one of their derivatives as an amino acid moiety served well as substrates for N-carbamoylsarcosine amidohydrolase. N-Carbamoylsarcosine, N-methyl-N-carbamoyl-D-alanine, N-carbamoylglycine, and N-carbamoyl-D-alanine were hydrolyzed at relative rates of 100, 12.8, 9.8, and 7.3, respectively, by the enzyme. N-Carbamoyl derivatives of D-tryptophan, D-phenylalanine, and those of some other amino acids including D-phenylglycine and p-hydroxy-D-phenylglycine were also hydrolyzed by the enzyme. For the L-isomers of all N-carbamoyl amino acids tested there was no production of ammonia, carbon dioxide, or the corresponding amino acids due to the action of the enzyme. Cupric, mercuric, and silver ions inhibited the enzyme strongly, and some thiol reagents were also found to be inhibitory.     

Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp.

The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp. anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources. Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate. ATP is cleaved into AMP and pyrophosphate. The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied. Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1). It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km. Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids. The first one is identical with benzoate-CoA ligase (E1). The second enzyme is a 2-aminobenzoate-CoA ligase (E2). It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates. The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase. The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U. Altenschmidt, C. Eckerskorn, and G. Fuchs, Eur. J. Biochem. 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.