Localization of arylsulfatase in Pseudomonas C12B.

Arylsulfatase was released almost completely from intact cells of Pseudomonas C12B after osmotic shock or after treatment with lysozyme. These results suggest that the enzyme is cell wall associated in this soil isolate.     

Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.     

The formation of choline O-sulphate by Pseudomonas C12B and other Pseudomonas species

Pseudomonas C(12)B and other Pseudomonas species released larger amounts of a (35)S-labelled metabolite into the medium when cultured on growth-limiting concentrations of Na(2)SO(4) as opposed to growth in SO(4) (2-)-sufficient media. The metabolite was found at all stages of the culture cycle of Pseudomonas C(12)B and maximum quantities occurred in stationary-phase culture supernatants. The metabolite was not detected when the bacterium was cultured on growth-limiting concentrations of potassium phosphate. The amount of the metabolite present in the medium greatly exceeded that which could be extracted from intact cells and, except for choline chloride, it was independent of the carbon source used for growth. If choline chloride was present in high concentration, then larger amounts of the metabolite were found in the culture medium. The metabolite was not detected extracellularly or intracellularly when the bacterium was grown in SO(4) (2-)-deficient media containing 5mm-l-cysteine. The same metabolite was also synthesized in vitro only when Pseudomonas C(12)B extracts were incubated with choline chloride, ATP, MgCl(2) and Na(2) (35)SO(4). The metabolite-forming system was not subject to repression by Na(2)SO(4) and was completely inhibited by 0.5mm-l-cysteine and activated by Na(2)SO(4) (up to 1.0mm). The metabolite was identified as choline O-sulphate by electrophoresis, chromatography and isotope-dilution analysis. Another (35)S-labelled metabolite was also detected in culture supernatants, but was not identified.     

Metabolic pathway for the biodegradation of sodium dodecyl sulfate by Pseudomonas sp. C12B

Metabolism of sodium dodecyl sulfate (SDS) by the detergent-degrading bacterium Pseudomonas C12B has been studied using a 14C radiotracer in combination with radio-respirometry, radio-TLC, and GLC. Metabolism was extensive with 70% of the radiolabel released as 14CO2 at completion. The remainder of the radiolabel was incorporated almost totally into cells. Ether extraction of cells indicated that 14C-labeled cellular material appearing early in the uptake process was predominantly ether-extractable (mainly 1-dodecanol) and was subsequently converted to more polar metabolites. Analysis of the extractable lipids established the sequential production from [1-14C]SDS of 1-dodecanol, dodecanal, and dodecanoic acid. At this point the pathway diverged leading either to formation of 14CO2 via beta-oxidation or to elongation to C14, C16, and C18 fatty acyl residues with rapid incorporation into lipid fractions such as phospholipids. The pathway was correlated with known long-chain alkylsulfatases and alcohol dehydrogenases in this isolate and indicated that hydrophobic metabolites of the alkyl chain of surfactants can be incorporated into cellular components such as membrane lipids without prior degradation by beta-oxidation.     

Localization of vitamin B12 binding in Euglena gracilis

Different fractionation procedures were used to determine the location of vitamin B12 binding sites in Euglena gracilis. Using uptake measurements, cell fractionation, and light and electron microscopy, the cuticle of the cell was found to be the fraction containing the majority of B12 binding sites. The apparent distribution of vitamin binding sites differed according to the cell lysis method used. The cuticle fraction was responsible for the binding of 80% of the vitamin taken up by the cell during both the rapid and the slow phase of uptake. These results suggest that vitamin B12 binding is regulated, in part, at the cuticle level, and support our previous conclusion that the secondary phase of uptake represents the synthesis of new receptor sites and not the unloading of vitamin inside the cell.     

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans.

Studies with cell-free protein preparations from a series of recombinant strains of Pseudomonas denitrificans demonstrated that precorrin-3 is converted into a further trimethylated intermediate, named precorrin-3B, along the pathway to coenzyme B12. It was then shown that the part of the pathway from precorrin-3 (called precorrin-3A hereafter) to precorrin-6x involves three intermediates, precorrin-3B, precorrin-4, and precorrin-5. Precorrin-3B was isolated in its native (reduced) as well as its oxidized (factor-IIIB) states, and precorrin-4 was isolated in its oxidized form only (factor-IV). Both factors were in vitro precursors of precorrin-6x. The synthesis of precorrin-6x from precorrin-3A was shown to be catalyzed by four enzymes, CobG, CobJ, CobM, and CobF, intervening in this order. They were purified to homogeneity. CobG, which converts precorrin-3A to precorrin-3B, was found to be an iron-sulfur protein responsible for the oxidation known to occur between precorrin-3A and precorrin-6x, and CobJ, CobM, and CobF are the C-17, C-11, and C-1 methylases, respectively. The acetate fragment is extruded after precorrin-4 formation. This study combined with our recent structural studies on factor-IV (D. Thibaut, L. Debussche, D. Fréchet, F. Herman, M. Vuilhorgne, and F. Blanche, J. Chem. Soc. Chem. Commun. 1993:513-515, 1993) and precorrin-3B (L. Debussche, D. Thibaut, M. Danzer, F. Debu, D. Fréchet, F. Herman, F. Blanche, and M. Vuilhorgne, J. Chem. Soc. Chem. Commun. 1993:1100-1103, 1993) provides a first step-by-step picture of the sequence of the enzymatic reactions leading to the corrin ring in P. denitrificans.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.     

Comparative biochemistry of murine arylsulfatase B

Arylsulfatase B was purified 4500-fold from liver and kidney of C57BL/6J mice. Hepatic and renal arysulfatase B are apparently determined by a single structural locus; however, posttranslational modification introduces inter- and intratissue microheterogeneity. Partially purified enzyme from C57BL/6J, A/J, C3H/HeJ, and SWR/J mice has similar catalytic properties. The 4500-fold-purified arylsulfatase B from SWR/J and C3H/HeJ mice was more thermostable than that from C57BL/6J and A/J mice, strongly suggesting that the thermostability difference reflects an alteration of the primary structure of the enzyme. Thermal stability of arylsulfatase B was pH dependent and markedly influenced by buffer anion. Variation of thermostability did not appear accountable for the observed activity variation among these strains; however, this possibility cannot be rigorously excluded by presently available data. Thirty-five murine strains were found to possess the As-1 ^a allele (thermostable enzyme), while As-1 ^b was largely restricted to A and C57 strains.This research was supported by PHS Biomedical Sciences Research Support Grant RR-07030.     

Alkane assimilation ability of Pseudomonas C12B originally isolated for degradation of alkyl sulfate surfactants

Summary Pseudomonas C12B (NCIMB 11753) is able to utilize a broad range of alkyl sulfates. The growth on n-alkanes of different chain lenght (C₆–C₁₆) was tested. Pseudomonas C12B assimilated hydrocarbons from C₉–C₁₆. Growth rate on n-decane (1%) that was chosen as the typical sole source of carbon and energy depended on oxygen supply. The addition of surfactants (Triton X-100 and Tween-80) in a nontoxic concentrations resulted in increased biomass yield. Under optimal growth conditions Pseudomonas C12B exhibited the maximal growth rate and yield with C₁₁ as the sole carbon source.     

Arylsulfatase from Pseudomonas sp. strain C12B: purification to homogeneity, immunological analysis, and physical properties.

Arylsulfatase was purified 219-fold from Pseudomonas sp. strain C12B. The final preparation was homogeneous by electrophoretic and immunological analysis. The enzyme is a monomer of molecular weight about 51,000, with a Stokes radius of 3.0 X 10(-7) cm, a frictional ratio of 1.2, and a sedimentation coefficient of 4.1S.     

Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans.

Cobalamin synthesis probably requires 20 to 30 different enzymatic steps. Pseudomonas putida and Agrobacterium tumefaciens mutants deficient in cobalamin synthesis (Cob have been isolated. In P. putida, Cob mutants were identified as being unable to use ethanolamine as a source of nitrogen in the absence of added cobalamin (deamination of ethanolamine requires coenzyme B12 as a cofactor). In A. tumefaciens, Cob mutants were simply screened for their reduced cobalamin synthesis. A genomic library of Pseudomonas denitrificans was constructed on a mobilizable wide-host-range vector. Eleven plasmids from this library were able to complement most of these mutants. By complementation and restriction mapping analysis, four genomic loci of P. denitrificans were found to be responsible for complementation of the Cob mutants. By subcloning fragments from the four genomic loci, we identified at least 14 different genes involved in cobalamin synthesis.     

Induction of primary alkylsulphatases and metabolism of sodium hexan-1-yl sulphate by Pseudomonas C12B

Sodium hexan-1-yl sulphate and certain related alkyl sulphate esters have been shown to serve as inducers of the formation of primary alkylsulphatases (designated as P1 and P2) in Pseudomonas C12B. When the organism is grown on sodium hexan-1-yl [(35)S]sulphate as the sole source of sulphur or as the sole source of carbon and sulphur only the P2 alkylsulphatase is formed and inorganic (35)SO(4) (2-) is liberated into the media. Cell extracts contain this anion as the major (35)S-labelled metabolite although two unidentified labelled metabolites as well as choline O-[(35)S]sulphate occur in trace quantities in some extracts. Dialysed cell extracts are capable of liberating inorganic (35)SO(4) (2-) from sodium hexan-1-yl [(35)S]sulphate without the need to include cofactors known to be required for the bacterial degradation of n-alkanes. The collective results suggest that sodium hexan-1-yl sulphate can act as an inducer of P1 alkylsulphatase formation without the need for prior metabolic modification of the carbon moiety of the ester.     

The metabolism of cyclopentanol by Pseudomonas N.C.I.B. 9872

1. Pseudomonas N.C.I.B. 9872 grown on cyclopentanol as carbon source oxidized it at a rate of 228mul of O(2)/h per mg dry wt. and the overall consumption of 5.9mumol of O(2)/mumol of substrate. Cyclopentanone was oxidized at a similar rate with the overall consumption of 5.2mumol of O(2)mumol of substrate. Cells grown with sodium acetate as sole source of carbon were incapable of significant immediate oxidation of these two substrates. 2. Disrupted cells catalysed the oxidation of cyclopentanol to cyclopentanone by the action of an NAD(+)-linked dehydrogenase with an alkaline pH optimum. 3. A cyclopentanolinduced cyclopentanone oxygenase (specific activity 0.11mumol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mumol of NADPH and 0.9mumol of O(2) in the presence of 1mumol of cyclopentanone. NADPH oxidation did not occur under anaerobic conditions. The only detectable reaction product with 100000g supernatant was 5-hydroxyvalerate. 4. Extracts of cyclopentanol-grown cells contained a lactone hydrolase (specific activity 7.0mumol hydrolysed/min per mg of protein) that converted 5-valerolactone into 5-hydroxyvalerate. 5. Cyclopentanone oxygenase fractions obtained from a DEAE-cellulose column were almost devoid of 5-valerolactone hydrolase and catalysed the formation of 5-valerolactone in high yield from cyclopentanone in the presence of NADPH. 6. Incubation of 5-hydroxyvalerate with the 100000g supernatant, NAD(+) and NADP(+) under aerobic conditions resulted in the consumption of O(2) and the conversion of 5-hydroxyvalerate into glutarate. 7. The high activity of isocitrate lyase in cyclopentanol-grown cells suggests that the further oxidation of glutarate proceeds through as yet uncharacterized reactions to acetyl-CoA. 8. The reaction sequence for the oxidation of cyclopentanol by Pseudomonas N.C.I.B. 9872 is: cyclopentanol --> cyclopentanone --> 5-valerolactone --> 5-hydroxyvalerate --> glutarate --> --> acetyl-CoA.     

The activity of arylsulfatase A and B on tyrosine O-sulfates.

L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.