Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3) from the hyperthermophilic Archeon Pyrococcus furiosus was purified to homogeneity by chromatography on anion-exchange, molecular-exclusion and hydrophobic-interaction media. The purified native enzyme had an M(r) of 270,000 +/- 15,000 and was shown to be a hexamer with identical subunits of M(r) 46,000. The enzyme was exceptionally thermostable, having a half-life of 3.5 to more than 10 h at 100 degrees C, depending on the concentration of enzyme. The Km of the enzyme for ammonia was high (9.5 mM), indicating that the enzyme is probably active in the deaminating, catabolic direction. The coenzyme utilization of the enzyme resembled the equivalent enzymes from eukaryotes rather than eubacteria, since both NADH and NADPH were recognized with high affinity. The enzyme displayed a preference for NADP+ over NAD+ that was more pronounced at low assay temperatures (50-70 degrees C) compared with the optimal temperature for enzyme activity, 95 degrees C.     

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pI of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100 degrees C was 12 h), totally independent of enzyme concentration. P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P. furiosus.     

Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a Mr of about 270,000 and was shown to be a hexamer of subunit Mr of 44,000. It was active from 30 to 95 degrees C, with a maximum activity at 85 degrees C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90 degrees C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.     

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.     

Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis

The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD+ as cofactors respectively. The enzyme showed low thermostability with T(m) = 41 degrees C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their T(m) values were 61 degrees C and 49 degrees C respectively. Furthermore, Q144R had a remarkably high k(cat) value (435 s(-1)) for amination reaction at 37 degrees C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.     

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80 C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Purification and properties of three NAD(P)+ isozymes of L-glutamate dehydrogenase of Chlamydomonas reinhardtii.

Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800-1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266,000-269,000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60-62 degrees C) and isoelectric points (7.9-8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5'-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentration-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13-53 mM; 0.36-1.85 mM for 2-oxoglutarate and 0.07-0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64-3.52 mM for L-glutamate and 0.20-0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperativity (n = 0.8).     

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.     

A Hyperactive NAD(P)H:Rubredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus

NAD(P)H:rubredoxin oxidoreductase (NROR) has been purified from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is exceedingly active in catalyzing the NADPH-dependent reduction of rubredoxin, a small (5.3-kDa) iron-containing redox protein that had previously been purified from this organism. The apparent Vmax at 80 degrees C is 20,000 micromol/min/mg, which corresponds to a kcat/Km value of 300,000 mM(-1) s(-1). The apparent Km values measured at 80 degrees C and pH 8.0 for rubredoxin, NADPH, and NADH were 50, 5, and 34 microM, respectively. The enzyme did not reduce P. furiosus ferredoxin. NROR is a monomer with a molecular mass of 45 kDa and contains one flavin adenine dinucleotide molecule per mole but lacks metals and inorganic sulfide. The possible physiological role of this hyperactive enzyme is discussed.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.