Optimum pH For Activity Research Articles

Biliverdin reductase from the liver of Atlantic salmon (Salmo salar).

Biliverdin reductase was characterized and purified from the liver of Atlantic salmon (Salmo salar) using a novel enzymatic staining method. The properties of the enzyme are quite different from those of mammals. The purified enzyme is a monomeric protein with a molecular weight of approximately 68 kD and an isoelectric point of around 3.8. The enzyme can utilize both NADH and NADPH as coenzyme, but the kinetic properties of the NADH-dependent and the NADPH-dependent enzyme activities are different: K(m) value for biliverdin IXalpha is 0.6 microM in the NADPH system, while it is 6.8 microM in the NADH system. Both enzyme activities are inhibited by excess biliverdin IXalpha, but the NADPH-dependent enzyme activity is far more susceptible. The optimum pH for activity is 5.5 with NADPH and 6.0 with NADH. The optimum reaction temperature is 35 degrees C.

Expression, purification, and characterization of 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase from carbazole-degrader Pseudomonas resinovorans strain CA10.

The two-subunit meta-cleavage enzyme, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked. SDS-PAGE and gel filtration showed that CarB was a alpha2beta2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb. The optimum pH for activity was 8.5 and that of temperature was 35 degrees C. The CarB enzyme had a Km of 14 microM and a kcat/Km of 0.25 microM(-1) s(-1) for 2'-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives. The enzyme was originally isolated as a meta-cleavage enzyme for 2'-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular β-glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for β-glucosidase A and β-glucosidase B, respectively. β-Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (β-glucosidase A, 29.2%; β-glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for β-glucosidases A and B, respectively. Both purified β-glucosidases. especially β-glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both β-glucosidases were highly activated by CuCl2, and inhibited by SnCl2 and KMnO4. Hg2+ and Ag+ also inhibited severely β-glucosidase B. The K m and V max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2μmol min−1 mg−1 for β-glucosidase A and 2.76 mM and 143.5 μmol min−1 mg−1 for β-glucosidase B.

KINETIC STUDIES OF AN AMYLASE USED COMMERCIALLY FOR THE CLARIFICATION OF APPLE JUICE

The effect of substrate and enzyme concentrations on the degradation of soluble corn starch with a commercial amylase (Rohalase HT) recommended for apple juice clarification was studied. The reaction was conducted at 50C at different pH values (3.6-5.6). Starch solutions containing from 2.48 to 14.85 mg/mL were used as substrate in the experiments. Influence of Ca 2+ on amylase activity was also determined. Obtained results showed that amylase exhibits an optimal activity pH value of 4.6, following a Michaelis-Menten type kinetics (K m = 8.11 mg/mL and ν max = 2.19 U/mg enzyme). The activity of amylase decreased significantly when CaCl 2 was added to the amylase solution (final amylase concentration in reaction mixture: 5-10 mM). An empirical equation relating the rate of product of hydrolysis formation (dC m /dt) with starch (C st ) and amylase (C e ) concentrations was obtained: dC m /dt = 1.18 × C 0.77 e × C1.03st × [1-exp(-0.11 × t)]. Cette etude evalue l'effet des concentrations en substrat et en enzyme sur la degradation d'amidon de mais soluble avec une amylase commerciale recommandee pour la clarification de jus de pomme. Les resultats indiquent que l'amylase a une activite optimale a pH 4,6, suit des cinetiques de type Michaelis-Menten (K m = 8.11 mg/mL et ν max = 2.19 U/mg enzyme). L'addition de CaCl 2 reduit significativement l'activite de l'enzyme.

Some biochemical properties of polyphenol oxidase from two varieties of avocado

A study of crude polyphenol oxidases from two avocado ( Persea americana Mill.) varieties, Booth 1 ( b1PPO ) and Julio Millán ( jmPPO ), were carried out to provide information useful for guiding food processing operations. Results are reported as “apparent” data because crude extracts were used, activities of which can be derived from a combination of isoenzymes. The pH-activity optimum was pH 7.5–7.6 for both extracts. Heat inactivation, between 67 and 84 °C was biphasic with activation energies ranging from 21.4 to 64.1 kcal/mol. The apparent substrate specificity was established from values V max/ K m as: 4-methyl catechol>chlorogenic acid>pyrogallol>catechol>caffeic acid> dl-DOPA for b1PPO. The substrate specificity for jmPPO was: 4-methyl catechol>chlorogenic acid>pyrogallol>caffeic acid>catechol> dl-DOPA>protocatechuic acid. The order of inhibitor effectiveness was: l-cysteine>ascorbic acid>resorcinol>glycine>NaCl.

Listeriolysin O: a genuine cytolysin optimized for an intracellular parasite.

Cholesterol-dependent cytolysins (CDCs)* are produced by a large number of pathogenic gram–positive bacteria. A member of this family, listeriolysin O (LLO), is produced by the intracellular pathogen Listeria monocytogenes. A unique feature of LLO is its low optimal pH activity (∼6) which permits escape of the bacterium from the phagosome into the host cell cytosol without damaging the plasma membrane of the infected cell. In a recent study (Glomski et al., 2002, this issue), Portnoy's group has addressed the molecular mechanism underlying the pH sensitivity of LLO. Unexpectedly, a single amino acid substitution in LLO L461T results in a molecule more active at neutral pH and promoting premature permeabilization of the infected cells, leading to attenuated virulence. This finding highlights how subtle changes in proteins can be exploited by bacterial pathogens to establish and maintain the integrity of their specific niches.

A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid.

An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity. It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor. The apparent K(m) value for nicotinic acid was determined to be 154 microM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2). NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite.

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0–9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20°C. The enzyme has a K m of 7.3 μM and a k cat of 21 s −1 for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25°C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10 5-fold lower activity than that of the wild-type enzyme.

Substrate specificity of arylamidase in soils

The enzyme amino acid arylamidase [EC 3.4.11.2] catalyzes the hydrolysis of an N-terminal amino acid from arylamides. Recently, this enzyme was detected in soils, and a method was developed for its assay. This method involves colorimetric determination of the β-naphthylamine produced when soil is incubated with L-leucine β-naphthylamide in 0.1 M THAM [tris(hydroxymethyl)aminomethane] buffer (pH 8.0) at 37°C for 1 h. Because this enzyme may be involved in N mineralization and because of the presence of a number of amino acids in soils, information on the substrate specificity of arylamidase in soils is needed for a better understanding of the reactions involved in organic N mineralization in soils. In this work eight compounds containing individual amino acid moieties linked to naphthylamine were studied as substrates for arylamidase activity in six surface soils. Results showed that the optimal pH of activity varied with the amino acid moiety, ranging from 7.0 to 9.0; most of the optimal pH values, however, were at 8.0. Among the amino acid moieties studied, the means of activity in six soils decreased with the following order: alanine>leucine>serine>lysine>glycine>arginine>histidine, and proline was not hydrolyzed in soils. The chromophore linked to the amino acids L-asparagine, L-tyrosine, L-glutamic acid, and L-aspartic acid did not dissolve. Calculated from arylamidase activity at the optimal pH values, the K m, V max, E a, and Δ H a values varied markedly among the soils and the amino acid moieties of the substrates studied. The means of the Q 10 values ranged from 1.04 to 1.66. Results suggest that there are more than two isoforms of this enzyme in soils, and that amino acid moieties of ring compounds in soils significantly affect the rate of N mineralization in soils.

Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3

In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P2 position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.

Characterization of polyphenoloxidase from medlar fruits ( Mespilus germanica L., Rosaceae)

Crude extracts prepared from medlar fruits ( Mespilus germanica L., Rosaceae) possess a diphenolase activity toward catechol, 4-methyl catechol, L-3,4-dihydroxyphenylalanine, epicatechin and 3-(3,4-dihydroxyphenyl)propionic acid. Among the substrates employed, the greatest substrate specificity was observed with 4-methylcatechol. The pH-activity optimum for the enzyme, in the presence of this substrate, was 6.5 and the pH-stability profile for the enzyme showed that 80% of the PPO activity was retained at physiological pH values. The temperature-activity optimum, for the enzyme in the presence 4-methyl catechol, was 35 °C. The enzyme was stable for 30 min at its optimum temperature and moderately stable at 60 °C. At higher temperatures, heat denaturation of the enzyme occurred after 10 min of incubation. Thermal inactivation parameters indicate that the medlar enzyme is very heat-labile. Moreover, the medlar PPO activity was very sensitive to some common PPO inhibitors, especially to cysteine and metabisulfite. All data indicate that medlar fruits have highly active PPO enzymes which possess similar biochemical and kinetic characteristics to other plant PPO enzymes.

A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences.

A highly alkaline endo-1,4-beta-glucanase (Egl) was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-N252. The optimal pH for activity was as high as 10, and the optimal temperature was 55 degrees C. The molecular mass and isoelectric point were around 50 kDa and pH 4.2, respectively. The enzyme hydrolyzed carboxymethyl cellulose in a random fashion. Unlike previously reported Egls, the enzyme was highly active on p-nitrophenyl cello-oligosaccharides and acid-swollen cellulose, and its activity was stimulated by cellobiose at high concentrations. The entire gene for the enzyme contained a 1,476-bp single open reading frame encoding 492 amino acids, including a 29-amino-acid signal peptide. The mature enzyme (463 amino acids: 51,174 Da) exhibited moderate homology to other family 5 alkaline Egls. In the C-terminal region, a carbohydrate-binding module that belongs to family XII was repeated. Furthermore, four and six repeats of Pro-Pro-Ser/Thr-Glu/Asp-Pro-(Glu) were found immediately before the first and second carbohydrate-binding modules, respectively.

Mutational analysis of the interaction between active site residues and the loop region in mammalian purple acid phosphatases.

Mammalian purple acid phosphatases (PAPs) can be divided into two groups, which exhibit distinct spectroscopic and kinetics properties: PAPs that consist of a single 36 kDa polypeptide, and PAPs that have undergone limited proteolysis to give two fragments with masses of 16 and 20 kDa, respectively. Proteolysis results in an increase in enzymatic activity, an increase in the optimal pH for activity, and a change in the g(z)() value of the characteristic EPR spectrum of the mixed-valence binuclear iron center. It has been proposed that these changes are due to the loss of interactions between Asp146 in an exposed loop region and active site residues upon proteolysis. In the present study, site-directed mutagenesis of Asp146 in recombinant rat bone PAP (recRPAP) has confirmed this hypothesis. Conversion of Asp146 into Ala, which eliminates the interaction of the side chain with the active site, resulted in an enzyme with properties typical of PAPs isolated in proteolytically cleaved forms. The Asp146Asn and Asp146Glu mutants were also prepared and examined to assess the effects of altered electrostatic interactions and side-chain length. Limited proteolysis of all three mutant enzymes with cathepsin L resulted in a significant increase in catalytic activity. Thus, although the interaction between Asp146 and (an) active site residue(s) is the major factor responsible for the low catalytic activity of uncleaved PAPs, other interactions are also important. Since both p-nitrophenyl phosphate and osteopontin, a potential in vivo substrate, show the same level of activation, the observed increase in catalytic activity upon proteolysis is likely to be due to electrostatic rather than steric effects. EPR spectra of FeZn-recRPAP before and after cleavage by cathepsin L suggest that cleavage primarily affects the divalent metal site. The observation that pK(es,1) is also sensitive to changes at the divalent site is consistent with the proposal that the nucleophilic hydroxide is that bridging the divalent and trivalent metals.

Human mitochondrial topoisomerase I.

Tension generated in the circular mitochondrial genome during replication and transcription points to the need for mtDNA topoisomerase activity. Here we report a 601-aa polypeptide highly homologous to nuclear topoisomerase I. The N-terminal domain of this novel topoisomerase contains a mitochondrial localization sequence and lacks a nuclear localization signal. Therefore, we refer to this polypeptide as top1mt. The pattern of top1mt expression matches the requirement for high mitochondrial activity in specific tissues. top1mt is a type IB topoisomerase that requires divalent metal (Ca(2+) or Mg(2+)) and alkaline pH for optimum activity. The TOP1mt gene is highly homologous to the nuclear TOP1 gene and consists of 14 exons. It is localized on human chromosome 8q24.3.

Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site.

We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40 kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. 'DNase I signature sequences', which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a 'somatomedin B motif' was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family.

Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site

We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. ‘DNase I signature sequences’, which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a ‘somatomedin B motif’ was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family.

Purification and characterization of a psychrophilic, calcium-induced, growth-phase-dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40 degrees C, and its activity decreased rapidly at 45 degrees C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

Stromal and Epithelial Expression of Tumor Markers Hyaluronic Acid and HYAL1 Hyaluronidase in Prostate Cancer

Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3-8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority ( approximately 75-80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3-8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0-4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a approximately 60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP.

7-Methylxanthine Methyltransferase of Coffee Plants: GENE ISOLATION AND ENZYMATIC PROPERTIES

Caffeine is synthesized through sequential three-step methylation of xanthine derivatives at positions 7-N, 3-N, and 1-N. However, controversy exists as to the number and properties of the methyltransferases involved. Using primers designed on the basis of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical proteins, a particular DNA fragment was amplified from an mRNA population of coffee plants. Subsequently, this fragment was used as a probe, and four independent clones were isolated from a cDNA library derived from coffee young leaves. Upon expression in Escherichia coli, one of them was found to encode a protein possessing 7-methylxanthine methyltransferase activity and was designated as CaMXMT. It consists of 378 amino acids with a relative molecular mass of 42.7 kDa and shows similarity to tea caffeine synthase (35.8%) and salicylic acid methyltransferase (34.1%). The bacterially expressed protein exhibited an optimal pH for activity ranging between 7 and 9 and methylated almost exclusively 7-methylxanthine with low activity toward paraxanthine, indicating a strict substrate specificity regarding the 3-N position of the purine ring. K(m) values were estimated to be 50 and 12 microM for 7-methylxanthine and S-adenosyl-l-methionine, respectively. Transcripts of CaMXMT could be shown to accumulate in young leaves and stems containing buds, and green fluorescent protein fusion protein assays indicated localization in cytoplasmic fractions. The results suggest that, in coffee plants, caffeine is synthesized through three independent methylation steps from xanthosine, in which CaMXMT catalyzes the second step to produce theobromine.

Flexibility with Truncation of the Amino- and Carboxyl-termini of Bacillus subtilis X-23 .ALPHA.-Amylase.

We analyzed the amino- and carboxyl-terminal amino acid sequences of five .α-amylases, Ba-L, Ba-S, pUXAI Amy, pUXA0 Amy, and pBLXA1 Amy, which originate from an α-amylase gene of Bacillus subtilis X-23. Ba-L and Ba-S were produced by wild-type strain B. subtilis X-23, while pUXAI Amy, pUXA0 Amy and pBLXA1 Amy were produced by Escherichia coli harboring dif ferent kinds of recombinant plasmids coding the α-amylase gene. Amino-terminal amino acid se quence analyses indicated that the signal peptides of Ba-L and Ba-S were the first 45 amino acid residues and those of pUXA1 Amy and pUXA0 Amy were the first 31 amino acid residues, while that of pBLXA1 Amy was the first 37 amino acid residues. Carboxyl-terminal sequence analyses showed that Ba-L and pUXA1 Amy contained the full length of the carboxyl-terminal-region polypeptide of the enzyme while Ba-S lacked 186 amino acid residues, and that pUXA0 Amy and pBLXA1 Amy lacked 88 and 189 amino acid residues, respectively, but had an extra 8 and 64 amino acid residues, respectively, originating from continuous translation of the vector region. All five enzymes showed the same characteristics with regard to molar activity, optimum temperature, optimum pH, and transglycosylation activity. These findings suggest that the amino- and carboxyl terminal regions of α-amylase have no effect on enzyme activity, but rather are flexible with regard to the truncation and addition of polypeptides.