Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans.

The cobalamin biosynthetic pathway enzyme that catalyzes amidation of 5'-deoxy-5'-adenosyl-cobyrinic acid a,c-diamide was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by a four-column procedure. The purified protein had an isoelectric point of 5.6 and molecular weights of 97,300 as estimated by gel filtration and 57,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. Stepwise Edman degradation provided the sequence of the first 16 amino acid residues at the N terminus. The enzyme catalyzed the four-step amidation sequence from cobyrinic acid a,c-diamide to cobyric acid via the formation of cobyrinic acid triamide, tetraamide, and pentaamide intermediates. The amidations are carried out in a specific order; this order was not determined. The enzyme was specific to coenzyme forms of substrates and did not carry out amidation of the carboxyl group at position f. The amidation reactions were ATP/Mg2+ dependent and exhibited a broad optimum around pH 7.5. L-Glutamine was shown to be the preferred amide group donor (Km congruent to 45 microM) but could be replaced by ammonia (Km = 20 mM). For all of the four partially amidated substrates, the Km values were in the micromolar range and the Vmax values were about 7,000 nmol h-1 mg-1.     

Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2

Cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium (CbiA) is the first glutamine amidotransferase in the anaerobic biosynthetic pathway of vitamin B(12) and catalyzes the ATP-dependent synthesis of cobyrinic acid a,c-diamide from cobyrinic acid using either glutamine or ammonia as the nitrogen source. The cbiA gene was cloned, the overexpressed protein was purified to homogeneity, and the kinetic parameters were determined. CbiA is a monomer with K(m) values of 0.74, 2.7, 53, and 26 200 microM for cobyrinic acid, ATP, glutamine, and ammonia, respectively. Analysis of the glutaminase partial reaction demonstrated that the hydrolysis of glutamine and the synthesis of the cobyrinic acid a,c-diamide product are uncoupled. The time course for the synthesis of the diamide product and positional isotope exchange experiments demonstrate that CbiA catalyzes the sequential amidation of the c- and a-carboxylate groups of cobyrinic acid via the formation of a phosphorylated intermediate. These results support a model for the catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate, and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate. Several conserved residues in the synthetase active site were mutated to address the molecular basis of the amidation order; however, no changes in the order of amidation were obtained. The mutants D45N, D48N, and E90Q have a dramatic effect on the catalytic activity, whereas no effect was found for the mutant D97N. The substitutions by alanine of L47 and Y46 residues specifically decrease the affinity of the enzyme for the c-monoamide intermediate.     

Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography

After initial pretreatment for removal of interfering substances, corrinoid precursors of cobalamin from cultures of Pseudomonas denitrificans were separated by HPLC with a gradient elution system. In this system, all the following compounds are separated in their dicyano form, and retention times are given: cobyrinic acid; cobyrinic acid a-amide; cobyrinic acid c-amide; cobyrinic acid g-amide; cobyrinic acid a,g-diamide; cobyrinic acid c,g-diamide; cobyrinic acid a,c-diamide; cobyrinic acid a,c,g-triamide; cobyrinic acid triamide, tetraamide, and pentaamide isolated from P. denitrificans; cobyric acid; cobinamide; cobinamide phosphate; GDP-cobinamide; cyanocobalamin 5'-phosphate; and cyanocobalamin. Application of this HPLC method to culture samples of P. denitrificans revealed that in this microorganism the level of cobyrinic acid and cobyrinic acid monoamide is far lower than that of all other corrinoid precursors of cobalamin and suggested that (i) the (R)-1-amino-2-propanol group is incorporated only after completion of all the other amidations and (ii) the amidations follow only one sequence. The usefulness of this HPLC method was further demonstrated by identifying the 57Co-labeled corrinoid precursors of cobalamin accumulated by cobalamin-deficient mutants of Agrobacterium tumefaciens. A TLC system that separates the different corrinoid intermediates (in their dicyano form) and cyanocobalamin is also described.     

Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.

Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.     

Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin

Co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli resulted in the production of cobyrinic acid a,c-diamide. A cbiD deletion mutant of this strain produced 1-desmethylcobyrinic acid a,c-diamide, indicating that CbiD is involved in C-1 methylation in the anaerobic pathway to cobalamin. Strains that did not have the cbiP gene also produced 1-desmethylcobyrinic acid a,c-diamide, and strains that had neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity.     

Neutral amino acid transport at the human blood-brain barrier

The kinetics of human blood-brain barrier neutral amino acid transport sites are described using isolated human brain capillaries as an in vitro model of the human blood-brain barrier. Kinetic parameters of transport (Km, Vmax, and KD) were determined for eight large neutral amino acids. Km values ranged from 0.30 +/- 0.08 microM for phenylalanine to 8.8 +/- 4.6 microM for valine. The amino acid analogs N-methylaminoisobutyric acid and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid were used as model substrates of the alanine- and leucine-preferring transport systems, respectively. Phenylalanine is transported solely by the L-system (which is sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and leucine is transported equally by the L- and ASC-system (which is sodium-dependent and N-methylaminoisobutyric acid-independent). Dose-dependent inhibition of the high affinity transport system by p-chloromercuribenzenesulfonic acid is demonstrated for phenylalanine, similar to the known sensitivity of blood-brain barrier transport in vivo. The Km values for the human brain capillary in vitro correlate significantly (r = 0.83, p less than 0.01) with the Km values for the rat brain capillary in vivo. The results show that the affinity of human blood-brain barrier neutral amino acid transport is very high, i.e. very low Km compared to plasma amino acid concentrations. This provides a physical basis for the selective vulnerability of the human brain to derangements in amino acid availability caused by a selective hyperaminoacidemia, e.g. hyperphenylalaninemia.     

Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin

Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp=N-[2,4-dinitrophenyl]-ethylenediamine and Abz=ortho-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).     

Metabolism of 2-oxoaldehyde in mold. Purification and characterization of two methylglyoxal reductases from Aspergillus niger

Two kinds of methylglyoxal reductases were purified to apparent homogeneity from Aspergillus niger and designated MGR I and MGR II. Both enzymes consisted of a single polypeptide chain with a relative molecular mass of 36,000 (MGR I) and 38,000 (MGR II). NADPH was specifically required for the activities of both enzymes and Km values for NADPH were 54 microM (MGR I) and 6.8 microM (MGR II). MGR I was specific to 2-oxoaldehydes [glyoxal, methylglyoxal (Km = 15.4 mM) and phenylglyoxal], whereas MGR II was active on both 2-oxoaldehydes [glyoxal (Km = 10 mM), methylglyoxal (Km = 1.43 mM), phenylglyoxal (Km = 4.35 mM) and 4,5-dioxovalerate] and some aldehydes (propionaldehyde and acetaldehyde). Optimal pH values for MGR I and MGR II activities were 9.0 and 6.5 respectively. Both enzymes were inactivated by a brief incubation with 2-oxoaldehydes (glyoxal, methylglyoxal and phenylglyoxal) in the absence of NADPH. MGR I activity was competitively inhibited by NADP+ and the Ki value for NADP+ was calculated to be 0.49 mM. On the other hand, the inhibition of MGR II activity by NADP+ was of mixed type, the Ki value for NADP+ being 45 microM. MGR I was different from MGR II in amino acid composition.     

Partial randomization of the four sequential amidation reactions catalyzed by cobyric acid synthetase with a single point mutation

Cobyric acid synthetase (CbiP) from Salmonella typhimurium catalyzes the glutamine and ATP-dependent amidation of carboxylates b, d, e, and g within adenosyl cobyrinic acid a,c-diamide. After each round of catalysis the partially amidated intermediates are released into solution and the four carboxylates are amidated in the sequential order of e, d, b, and g for the wild type enzyme. In the presence of [gamma-18O4]-ATP and adenosyl cobyrinic a,c-diamide the enzyme will catalyze the positional isotope exchange of the betagamma-bridge oxygen with the two beta-nonbridge oxygens. These results support the proposal that ATP is used to activate the carboxylate groups via the formation of a phosphorylated intermediate. CbiP catalyzes the hydrolysis of glutamine in the absence of ATP or adenosyl cobyrinic acid a,c-diamide, but the rate of glutamine hydrolysis is enhanced by a factor of 60 in the presence of these two substrates together. This result suggests that the formation of the phosphorylated intermediate is coupled to the activation of the site utilized for the hydrolysis of glutamine. However, the rate of glutamine hydrolysis is approximately 2.5 times the rate of ADP formation, indicating that the two active sites are partially uncoupled from one another and that some of the ammonia from glutamine hydrolysis leaks into the bulk solution. The mutation of D146 to either alanine or asparagine results in a protein that is able to catalyze the formation of cobyric acid. However, the strict amidation order observed with the wild type CbiP is partially randomized with carboxylate b being amidated last. With the D146N mutant, the predominant pathway occurs in the sequence d, e, g, and b. It is proposed that this residue enforces the amidation order in the wild type enzyme via charge-charge repulsion between the side chain carboxylate and the carboxylates of the substrate.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Purification and characterization of trypsin from the poikilotherm Gadus morhua

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.     

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.     

Active transport of nonpolar amino acids in Chromatium vinosum

The photosynthetic purple sulfur bacterium, Chromatium vinosum, takes up the amino acids, L-phenylalanine and L-leucine, via two apparently different electrogenic, H+/amino acid symports. Na+ serves as an allosteric modulator for leucine transport, lowering the Km for leucine from 66 to 15 microM. C. vinosum cells also contain a system that transports both isoleucine and valine. The isoleucine/valine system has the attributes of a H+/amino acid symport at pH less than 7.5 but appears to function as a H+/Na+ (Li+)/amino acid symport at pH greater than or equal to 7.5. Na+ gradients produce an allosteric lowering of the Km values for both isoleucine and valine, from 14 to 7 microM and from 34 to 17 microM, respectively. C. vinosum also accumulates D-alanine in an energy-dependent reaction. The transport process appears to involve the electrogenic cotransport of D-alanine and Na+. The Km value for D-alanine was determined to be 9 microM. Unlike the previously characterized C. vinosum L-alanine/Na+ symport, Na+ gradients did not affect the Km for D-alanine transport. L-Alanine and glycine, but not alpha-aminoisobutyric acid, act as competitive inhibitors for D-alanine transport.     

Long-chain-acyl-CoA synthetase and very-long-chain-acyl-CoA synthetase activities in peroxisomes and microsomes from rat liver. An enzymological study

We have investigated the palmitic acid (C16:0) and cerotic acid (C26:0) activating activities in rat-liver microsomes and peroxisomes. The activation of the two fatty acids showed similar dependencies on ATP and coenzyme A, reflected in about equal apparent Km values both in microsomes and peroxisomes. In microsomes and peroxisomes similar apparent Km values for palmitic acid were found (15 microM and 22.8 microM, respectively), whereas apparent Km values for cerotic acid were 8.4 microM and 1.0 microM in microsomes and peroxisomes, respectively. The activation of cerotic acid was found to be inhibited to a progressively greater extent by increasing concentrations of 1-pyrenedecanoic acid (P10) as compared to the activation of palmitic acid, both in microsomes and peroxisomes. The inhibition by P10 of palmitic acid activation and cerotic acid activation was non-competitive in both organelles. From the observation that P10 activation is not affected by palmitic acid and cerotic acid, we conclude that P10 is activated by a distinct enzyme. Furthermore, our results are in accordance with earlier suggestions that activation of cerotic acid is brought about by an enzyme distinct from the palmitoyl-CoA synthetase.     

Difference in sodium requirement of branched chain amino acid carrier between Pseudomonas aeruginosa PAO and PML strains is due to substitution of an amino acid at position 292

Sodium dependence of leucine transport, a measure of the Na+-coupled leucine-isoleucine-valine II (LIV-II) transport system in Pseudomonas aeruginosa, was compared between two wild-type strains, PAO and PML. The leucine transport activity was saturated at 0.1 mM NaCl for PAO and at 5.0 mM for PML. From kinetics experiments, the apparent Km value for Na+ with respect to leucine transport was estimated to be 3 microM for PAO and 95 microM for PML. The Km value for leucine was 6 microM for PAO and 13 microM for PML. The LIV-II carrier gene (braB) of PML was isolated for comparison of its amino acid sequence with that of the PAO carrier cloned previously. The Km values for Na+ and leucine of the cloned LIV-II carriers of PAO and PML expressed in LIV-II defective mutants were similar to those in wild-type strains. Determination of the nucleotide and deduced amino acid sequences of the LIV-II carrier gene of PML showed an amino acid difference at position 292 between the PAO and PML carriers. The amino acid was threonine for PAO and alanine for PML. These results indicate that the substitution of the amino acid at position 292 of the LIV-II carrier causes a difference in the sodium requirement of the carriers of the PAO and PML strains.     

Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase.

A 5.4-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment carries five different cob genes (cobA to cobE). Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT. For the same reasons the cobB gene was shown to be the structural gene for cobyrinic acid a,c-diamide synthase. Genetic and biochemical data concerning cobC and cobD mutants suggest that the products of these genes are involved in the conversion of cobyric acid to cobinamide.     

Enzymes of vitamin B6 degradation. Purification and properties of isopyridoxal dehydrogenase and 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic-acid dehydrogenase

Two NAD+-dependent, highly specific pyridine-5-aldehyde dehydrogenases, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic-acid (Compound 1) dehydrogenase and isopyridoxal dehydrogenase, were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. Both enzymes are induced in response to growth of the organisms on pyridoxine and catalyze steps in the degradation of this compound by these organisms. Compound 1 dehydrogenase (Mr = 65,000) contains two subunits of equal size with methionine as the NH2-terminal amino acid and acts optimally at pH 7.8-8.5. It catalyzes with equal facility (turnover number = 400-670 s-1 molecule-1) both the oxidation of Compound 1 (Km = 65 microM) by NAD+ (Km = 25 microM) to 3-hydroxy-2-methylpyridine-4,5-dicarboxylic acid and the reduction of Compound 1 by NADH (Km = 20 microM) to 4-pyridoxic acid and appears to act as a true dismutase. The possible advantage to the organism of its ability to act as a dismutase is discussed briefly. No oxidation of 4-pyridoxic acid by this enzyme was observed. Isopyridoxal dehydrogenase (Mr = 242,000) contains four subunits of equal size, again with methionine at the NH2 terminus. At its optimal pH of 8.0-8.6, it catalyzes the oxidation of isopyridoxal (Km = 40 microM, turnover number = 10 s-1 molecule-1) by NAD+ (Km = 40 microM) to a mixture of 5-pyridoxic acid and 5-pyridoxolactone, which are produced in constant ratio throughout the course of the reaction. Formation of the two products, although unusual, is readily understandable in terms of the structure of isopyridoxal in solution or the structure of a possible acyl-enzyme intermediate in the oxidative reaction.