Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter⁻¹. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter⁻¹ after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter⁻¹, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter⁻¹. The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

Inhibition of Zea mays coniferyl aldehyde dehydrogenase by daidzin: A potential approach for the investigation of lignocellulose recalcitrance

Coniferyl aldehyde dehydrogenase (CALDH) catalyzes the oxidation of coniferyl aldehyde to ferulic acid. Because ferulic acid has a relevant role in the structure and recalcitrance of the cell wall, inhibition of CALDH can reduce its levels and increase the digestibility of lignocellulosic biomass. We prospected in silico a selective inhibitor of CALDH of Zea mays. The ZmaysCALDH gene was identified by homology with the corresponding gene of Arabidopsis thaliana. The sequence was translated and analyzed, and the quaternary structure was modeled. A set of 20 putative inhibitors were screened from a virtual library and docked in the active site of ZmaysCALDH, and daidzin (DZN) was selected as an enzyme inhibitor. The stability of the ZmaysCALDH–DZN complex was evaluated by molecular dynamics simulations of the monomeric and tetrameric forms. For evaluation of kinetic analysis, ZmaysCALDH activity was determined in vitro by high-performance liquid chromatography. In comparison to the DZN-free control, the data obtained indicated constant V_max and enhanced K_m. Altogether, in silico and in vitro findings indicated that DZN inhibited ZmaysCALDH competitively. The DZN-induced inhibition of ZmaysCALDH could be a valuable and promising approach to studies on ferulic acid biosynthesis and saccharification of lignocellulosic biomass.     

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)₈-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp²⁴¹, Glu²⁹⁵, Asp³⁶⁹, His¹⁴⁵, and His³⁶⁸) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a K_m of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe³⁺ and Hg²⁺ and was enhanced by Mn²⁺ and Mg²⁺. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu⁴⁹⁸ and Arg³¹⁰ with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.     

Biochemical and Genetic Analyses of Ferulic Acid Catabolism in Pseudomonas sp. Strain HR199

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding β-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsΩGm and Pseudomonas sp. strain HRechΩKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatΩKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a β-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.     

Identification and Characterization of a Pseudomonas Strain Capable of Metabolizing Phenoxybenzoates

3-Phenoxybenzoate is a transient metabolite from the breakdown of a number of pyrethroid insecticides in soil. In this study, we identified and characterized a bacterium which could grow on 3-phenoxybenzoate, converting it to phenol. On the basis of morphological and biochemical features, the 3-phenoxybenzoatedegrading isolate was determined to be a Pseudomonas species, probably a strain of Pseudomonas delafieldii, now designated Pseudomonas strain ET1. Pseudomonas strain ET1 grew on 3-phenoxybenzoate with a generation time of 3 h and a specific rate of metabolism of (2.6 ± 0.9) × 10^-13 g of 3-phenoxybenzoate consumed cell^-1 h^-1. The K_m for 3-phenoxybenzoate metabolism was 1.4 ± 0.8 μM. The metabolism of 3-phenoxybenzoate was constitutive and not subject to catabolite repression. The metabolism of a variety of substituted diaryl ether compounds was examined. 3- and 4-Phenoxybenzoates were metabolized, but 2-phenoxybenzoate was not. Phenoxy-substituted benzyl aldehyde was metabolized, but phenoxy-substituted benzyl alcohol, benzene, phenol, and aniline were not. Derivatives of 3-phenoxybenzoate substituted in the 4′ position with hydroxyl, methyl, or chlorine were metabolized, yielding the corresponding 4-substituted phenol. 3-(2-Hydroxyphenoxy)benzoate was not metabolized, but 3-phenoxy-4-fluorobenzoate was. These results indicate that the metabolism of the tested diaryl ether compounds was restricted to 4-phenoxybenzoate, 3-phenoxybenzyl aldehyde, and 3-phenoxybenzoate derivatives without a substitution in the 2′ position.     

Partial Purification and Characterization of the Maize Mitochondrial Pyruvate Dehydrogenase Complex

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1β-, E1α-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg²⁺. Michaelis constants for the substrates were 47, 3, 77, and 1 μm for pyruvate, coenzyme A (CoA), NAD⁺, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD⁺ and CoA, and the inhibition constants were 15 and 47 μm, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg²⁺.     

Purification and Characterization of Monovalent Cation-Activated Levodione Reductase from Corynebacterium aquaticum M-13

(6R)-2,2,6-Trimethyl-1,4-cyclohexanedione (levodione) reductase was isolated from a cell extract of the soil isolate Corynebacterium aquaticum M-13. This enzyme catalyzed regio- and stereoselective reduction of levodione to (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol). The relative molecular mass of the enzyme was estimated to be 142,000 Da by high-performance gel permeation chromatography and 36,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required NAD⁺ or NADH as a cofactor, and it catalyzed reversible oxidoreduction between actinol and levodione. The enzyme was highly activated by monovalent cations, such as K⁺, Na⁺, and NH₄⁺. The NH₂-terminal and partial amino acid sequences of the enzyme showed that it belongs to the short-chain alcohol dehydrogenase/reductase family. This is the first report of levodione reductase.     

Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD⁺-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (ΩKm) or gentamycin (ΩGm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3′-O-phosphotransferase gene and the gentamycin- 3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by ΩKm or ΩGm, and the functional vdh gene was replaced by vdhΩKm or vdhΩGm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HRvdhΩKm, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase. Received: 19 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

Biotransformation of Eugenol to Ferulic Acid by a Recombinant Strain of Ralstonia eutropha H16

The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.     

Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

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

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C₅ acyl side chain of the C₂₄ steroid compound cholate involves the C₂₂ intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C₂₄ steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD⁺ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.     

Isolation and Characterization of a Xylanase from Bacillus subtilis

Partial characterization of an extracellular xylanase isolated by chromatography from Bacillus subtilis gave a molecular weight of 32,000 and optimum pH and temperature of 5.0 and 50°C, respectively. K_m and V_max values, determined with a soluble larchwood xylan, were 0.16% and 7.0 × 10³ μmol min⁻¹ mg⁻¹ of enzyme respectively. The amino acid composition showed more basic amino acid residues than in a previously characterized xylanase from a white-rot fungus.     

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

Two different Cd²⁺ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn²⁺ uptake system which also takes up Cd²⁺ and is induced by Mn²⁺ starvation. The calculated K_m and V_max are 0.26 μM and 3.6 μmol g of dry cell⁻¹ min⁻¹, respectively. Unlike Mn²⁺ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd²⁺ uptake is weakly inhibited by citrate. Cd²⁺ and Mn²⁺ are competitive inhibitors of each other, and the affinity of the system for Cd²⁺ is higher than that for Mn²⁺. The other Cd²⁺ uptake system is expressed in Mn²⁺-sufficient cells, and no K_m can be calculated for it because uptake is nonsaturable. Mn²⁺ does not compete for transport through this system, nor does any other tested cation, i.e., Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, or Ni²⁺. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn²⁺-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn²⁺ for growth as the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn²⁺ and Cd²⁺ uptake system.     

Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium Methylocystis sp. Strain WI 14

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α₂β₂γ₂ structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg²⁺ ions (with a total loss of enzyme activity at 0.01 mM Hg²⁺) and Cu²⁺, Zn²⁺, and Ni²⁺ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).     

A Cold-Adapted Lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C₄ and C₆) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The K_m of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn²⁺, Cu²⁺, Fe³⁺, and Hg²⁺ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.     

A Membrane-Bound NAD(P)+-Reducing Hydrogenase Provides Reduced Pyridine Nucleotides during Citrate Fermentation by Klebsiella pneumoniae

During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H₂/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO₂. Upon addition of NiCl₂ (36 μM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H₂-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)⁺ was also achieved by the addition of formate. In crude extracts, the H₂:NAD⁺ oxidoreductase activity was 0.13 μmol min⁻¹ mg⁻¹, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 μmol of NADPH formed min⁻¹ mg⁻¹ at pH 7.0 and 1.7 μmol of NADH formed min⁻¹ mg⁻¹ at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na⁺/H⁺ antiporter monensin, the H₂-dependent reduction of NAD⁺ by membrane vesicles decreased only slightly (about 16%). The NADP⁺- or NAD⁺-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H₂ as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H₂:NAD(P)⁺ oxidoreductase that provides reducing equivalents for the synthesis of cell material.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

NADH Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP): Applications to Enzyme and Mitochondrial Reaction Kinetics, In Vitro

NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) was evaluated for studying enzyme kinetics in vitro and in isolated mitochondria. Mass, optical, and nuclear magnetic resonance spectroscopy data were consistent with the UV NADH photolysis reaction being NADH → NAD^· + H⁺ + e⁻. The overall net reaction was O₂ + 2NADH + 2H⁺ → 2NAD⁺ + 2H₂O, or in the presence of other competing electron acceptors such as cytochrome c, NADH + 2Cyt_ox → NAD⁺ + H⁺ + 2Cyt_red. Solution pH could differentiate between these free-radical scavenging pathways. These net reactions represent the photooxidation of NADH to NAD⁺. Kinetic models and acquisition schemes were developed, varying [NADH] and [NAD] by altering NADH photolysis levels, for extracting kinetic parameters. UV irradiation levels used did not damage mitochondrial function or enzymatic activity. In mitochondria, [NADH] is a high affinity product inhibitor that significantly reduced the NADH regeneration rate. Matrix NADH regeneration only slightly exceeded the net rate of NADH consumption, suggesting that the NADH regeneration process is far from equilibrium. Evaluation of NADH regeneration in active mitochondria, in comparison to rotenone-treated preparations, revealed other regulatory elements in addition to matrix [NADH] and [NAD] that have yet to be fully characterized. These studies demonstrate that the rapid UV photolysis of NADH to NAD is an effective tool in evaluating the steady-state kinetic properties of enzyme systems. Initial data support the notion that the NADH regeneration process is far from equilibrium in mitochondria and is potentially controlled by NADH levels as well as several other matrix factors.