ywfE in Bacillus subtilis codes for a novel enzyme, L-amino acid ligase

The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and gamma-peptide, but, curiously, no enzyme synthesizing alpha-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of alpha-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.     

Immobilization of β-d-galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: characterization and lactose hydrolysis

β-D-Galactosidase (BGAL) from Kluyveromyces lactis was covalently immobilized to functionalized silicon dioxide nanoparticles (10-20 nm). The binding of the enzyme to the nanoparticles was confirmed by Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Functionalized nanoparticles showed 87% immobilization yield. Soluble and immobilized enzyme preparation exhibited pH-optima at pH 6.5 and 7.0, respectively, with temperature optima at 35 and 40°C, respectively. Michaelis constant (K(m)) was 4.77 and 8.4mM for free and immobilized BGAL, respectively. V(max) for the soluble and immobilized enzyme was 12.25 and 13.51 U/ml, respectively. Nanoparticle immobilized BGAL demonstrated improved stability after favoring multipoint covalent attachment. Thermal stability of the immobilized enzyme was enhanced at 40, 50 and 65°C. Immobilized nanoparticle-enzyme conjugate retained more than 50% enzyme activity up to the eleventh cycle. Maximum lactose hydrolysis by immobilized BGAL was achieved at 8h.     

Characterization and phylogenetic analysis of a thermostable N-carbamoyl-<Emphasis Type="SmallCaps">l</Emphasis>-amino acid amidohydrolase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC11223

A thermostable N-carbamoyl- l-amino acid amidohydrolase ( l-N-carbamoylase) gene composed of an 1,230-bp ORF encoding a 44.3-kDa protein was cloned from the thermophile Bacillus kaustophilus CCRC11223. This l-N-carbamoylase contained six cysteine residues that form three disulfide bridges. The purified l-N-carbamoylase was stringently l-specific and exhibited high activity in the hydrolysis of N-carbamoyl- l-homophenylalanine. N-carbamoyl derivatives of beta-alanine, beta-aminoisobutyric acids, l-tryptophan, and d-specific amino acids were not recognized as substrates. The l-N-carbamoylase required the divalent metal ions Mn(2+), Co(2+), and Ni(2+) for increasing activity. The pH and temperature optima of the enzyme were pH 7.4 and 70 degrees C, respectively. This enzyme was completely thermostable at 50 degrees C for 36 days in the presence of d- and/or l-specific substrates. Phylogenetic analysis of the available amino acid sequences of N-carbamoyl and N-acyl amino acid amidohydrolases from the three main kingdoms of life showed that they can be divided into four distinct families. The B. kaustophilus enzyme could be classified into the family of l-N-carbamoylases and some beta-ureidopropionases, but did not hydrolyze beta-ureidopropionates.     

蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C(Phospholipase C,PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B.cereus基因组DNA为模板,PCR扩增得到磷脂酶C基因(bcplc),构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中,实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C(rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C,纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg,最适反应温度为80°C,最适pH为9.0。在低于40°C时,pH 7.0-8.0时,rbcPLC重组酶较稳定。Cu~(2+)和Co~(2+)对其有明显的抑制作用;Zn~(2+)、Mn~(2+)、Ca~(2+)、Mg~(2+)对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析,为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1

A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).     

The use of crude lipase in deprotection of C-terminal protecting groups

A crude lipase, Newlase F, was used to remove C-terminal protecting groups from dipeptide esters. Hydrolysis of dipeptide n-heptyl esters with Newlase F was conducted in aqueous media containing acetonitrile. The optimum pH and temperature of lipase in Newlase F were 7.0 and 30 °C, respectively. Low level acetonitrile promoted the hydrolysis of dipeptide n-heptyl esters, while high level acetonitrile inhibited the hydrolysis. However, the protease activity in Newlase F was significantly inhibited by acetonitrile. Lipase in Newlase F worked better in a medium containing water-miscible organic solvents than in water-immiscible ones. N-terminal protecting groups were not affected by the protease in the crude enzyme. It was found that the protease in Newlase F did not hydrolyze amide bond with hydrophilic amino acids on either side under these conditions (pH 7.0, room temperature). Newlase F may consequently be used widely in the synthesis of peptide conjugates. The crude enzyme was immobilized on SBA-15 mesoporous molecular sieve. The lipase activity of immobilized preparation was more active on hydrolysis of C-terminal protecting groups and stable than the free enzyme. The immobilization also reduced the protease activity.     

Purification and Characterization of a Novel Cholesterol Esterase from Pseudomonas aeruginosa,with Its Application to Cleaning Lipid-stained Contact Lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53°C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25°C, and retained its activity up to 53°C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate>oleate>stearate>palmitate>caprylate>myristate> laurate, caprate>caproate>butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

Some properties of cathepsins chemically fixed to carriers.

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.     

Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

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+.     

Catalytic properties of sepharose-bound l-Alanine dehydrogenase from Bacillus cereus

(1) l-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, l-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH₄⁺, pyruvate and l-alanine, was not different to that of the soluble form. The K_m of the Sepharose-bound l-alanine dehydrogenase for pyridine coenzymes was 6–8-times higher than in the soluble case. (3) The stability of l-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37°C for 24 h of the immobilized l-alanine dehydrogenase with 3 M NH₄Cl/NH₄OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the ‘solubilized’ l-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble l-alanine dehydrogenase.     

The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.     

A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein

A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.     

Purification and properties of aminopeptidase C from porcine skeletal muscle.

1. Aminopeptidase C was purified from porcine skeletal muscle. 2. The mol. wt of the enzyme was found to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. 3. The optimum pH for the hydrolysis of L-leucine p-nitroanilide was around 7.0. 4. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. 5. The enzyme acted on the beta-naphthylamide derivatives of amino acids and oligopeptides.     

Sinorhizobium morelens S-5中N-氨甲酰基水解酶基因的克隆、表达和纯化

N-氨甲酰基水解酶是一种非常具有工业应用价值的水解酶,可用于制备光学纯氨基酸。通过LA PCR从Sinorhizobium morelensS-5菌中克隆到1.3kb的DNA片段,测序表明该片段上含有一个完整的N-氨甲酰基水解酶的基因(hyuC)序列。将hyuC基因克隆到表达载体pET30a上,重组质粒pET30a-HyuC在大肠杆菌中获得了高水平表达。重组的N-氨甲酰基水解酶经过热处理和三步柱色谱分离而纯化。纯化倍数为16.1倍,收率21.2%。该酶为同源四聚体,亚基分子量是38kDa。最适温度是60℃,最适pH为7.0。该酶有较高的热稳定性和氧化稳定性。Fe2 和Ca2 对酶的活性有一定的促进作用,而金属螯合剂和巯基试剂对酶活无明显影响。     

Phosphoenolpyruvate carboxylase from the roots of yellow lupin (Lupinus luteus)

A crude preparation of PEP carboxylase (EC 4.1.1.31) from the yellow lupin roots exhibits the pH optimum of activity within the range of 7.4-8.6 and the temperature optimum at 32 - 40 degrees C. Its Km for PEP is 0.1 mM, and Km for HCO3- is 0.7 mM. The affinity of the enzyme towards Mg2+ diminishes with the metal ion concentration. At the concentration of Mg2+ below 0.5 mM Km for Mg2+ is 0.07 mM and at the Mg2+ concentration over 1.5 mM it rises to 0.47 mM. The Hill coefficients are 0.37 and 0.88, respectively. Among several compounds affecting the PEP carboxylase activity, such as organic acids, amino acids, and sugar phosphates, at physiological pH (7.0 and 7.8), malate shows the strongest inhibition of a competitive character, its Ki being 2 mM. Also acidic amino acids strongly inhibit the enzyme activity, aspartate being more effective than glutamate. Glucose 6-phosphate and fructose 1,6-diphosphate markedly activate the enzyme. Both the inhibition by malate, aspartate and glutamate, and the activation by sugar phosphates rises considerably when pH is decreased from 7.8 to 7.0. Malonate scarcely affects the enzyme.     

Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis

The beta-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH(2)-terminal amino acid sequence of the purified enzyme indicate that the beta-galactosidase subunit is composed of 1,038 amino acids with a calculated M(r) of 118,068. This beta-galactosidase shares structural properties with Escherichia coli beta-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis beta-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant beta-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis beta-galactosidase can outperform the current commercial beta-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted beta-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae)

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.