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Specificity of P2 primary alkylsulphohydrolase induction in the detergent-degrading bacterium Pseudomonas C12B. Effects of alkanesulphonates, alkyl sulphates and other related compounds.

ABSTRACT: Primary alkanesulphonates were shown to serve as non-metabolizable (gratuitous) inducers of the P2 primary alkylsulphohydrolase enzyme in resting cell suspensions of Pseudomonas C12B. The effects of increasing concentrations of inducer on the production of enzyme were complex and suggestive of a multiphasic phenomenon. However, it was possible to determine Kinducer constants (analogous to Km or Ki) for alkanesulphonates of chain length from C7 to c12. these decreased with increasing chain length in a manner characteristic of an homologous series. Primary alkyl sulphates also served as good inducers of alkylsulphohydrolase, but valid kinetic values could not be obtained because these esters are good substrates for the enzyme and are therefore appreciably hydrolysed during the induction period. Small amounts of enzyme were also produced when cyprinol sulphate, dodecyltriethoxy sulphate C12H23-[O-CH2-CH2]3-O-SO3-Na+), Crag herbicide and some secondary alkyl sulphates were tested as inducers.

SUBMITTER: Cloves JM

PROVIDER: S-EPMC1161264 | biostudies-other | 1980 Jan

REPOSITORIES: biostudies-other

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Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B.

Project description:The P2 primary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200-250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a mol. wt. of 160 000. Activity towards primary alkyl sulphate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulphate substrates (C6-C12), logKm decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5kJ/mol for each additional CH2 group in the alkyl chain. logKi for the competitive inhibition by secondary alkyl 2-sulphate esters followed a similar pattern (-2.4kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. logKi values for inhibition primary alkanesulphonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accomodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulphate group, were not detectable at the active site.

| S-EPMC1161265 | biostudies-other

Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B.

Project description:The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000--46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C--O bond of the C--O--S ester linkage was confirmed by 18O-incorporation studies.

| S-EPMC1162506 | biostudies-other

Purification and properties of the S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B.

Project description:The S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B, was separated from other alkylsulphohydrolases and purified to homogeneity. Under the experimental conditions used the enzyme completely hydrolysed d-octan-2-yl sulphate (d-1-methylheptyl sulphate), but showed no activity towards the corresponding l-isomer. Additional evidence has been obtained to indicate that it is probably optically stereospecific for d-secondary alkyl sulphate esters with the ester sulphate group at C-2 and with a chain length of at least seven carbon atoms. Enzyme activity towards racemic samples of heptan-2-yl sulphate (1-methylhexyl sulphate), octan-2-yl sulphate and decan-2-yl sulphate (1-methylnonyl sulphate) increased with increasing chain length. l-Octan-2-yl sulphate is a competitive inhibitor of the enzyme, as are certain primary alkyl sulphates and primary alkanesulphonates. Inhibition by each of the last two types of compounds is characteristic of the behaviour of an homologous series. Inhibition increases with increasing chain length and plots of log K(i) values against the number of carbon atoms in each alkyl chain show the expected linear relationship. A crude preparation of the S2 secondary alkylsulphohydrolase was used to show that this particular enzyme hydrolyses l-octan-2-yl sulphate, but is probably inactive towards the corresponding d-isomer. The similarity of the S1 and S2 enzymes to the CS2 and CS1 enzymes respectively of Comamonas terrigena was established, and some comments have been made on the possible roles of these and other alkylsulphohydrolases in the biodegradation of detergents.

| S-EPMC1183839 | biostudies-other

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

Project description: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.

| S-EPMC1146854 | biostudies-other

The mechanism of action of primary alkylsulphohydrolase and arylsulphohydrolase from a detergent-degrading micro-organism.

Project description:Previous studies have shown that secondary alkylsulphohydrolases from certain detergent-degrading micro-organisms are unusual esterases in that they catalyse fission of the C-O bond of the alkyl sulphate ester linkage. The position of bond fission catalysed by a primary alkylsulphatase and an arylsulphohydrolase present in Pseudomonas C12B has now been investigated. The primary alkylsulphatase behaved like the secondary alkylsulphohydrolases in cleaving the C-O bond of potassium heptan-1-yl sulphate. In contrast, the arylsulphohydrolase, in common with other similar enzymes previously studied, catalysed the fission of the O-S bond of potassium p-nitrophenyl sulphate.

| S-EPMC1183735 | biostudies-other

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter-Alkyl-Chain Distance and Alkyl Chain Length.

Project description:Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile-lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins.

| S-EPMC5500196 | biostudies-literature

Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds.

Project description:Oligonucleotide-directed mutagenesis has been used to replace arginine-47 with glutamate in cytochrome P-450 BM3 from Bacillus megaterium and in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measurements of substrate binding. The mutant retains significant catalytic activity towards C12-C16 fatty acids, catalysing hydroxylation in the same (omega-1, omega-2, omega-3) positions with kcat/Km values a factor of 14-21 lower. C12-C16 alkyl trimethylammonium compounds are relatively poor substrates for the wild-type enzyme, but are efficiently hydroxylated by the arginine-47-->glutamate mutant at the omega-1, omega-2 and omega-3 positions, with kcat values of up to 19 s-1. Optical spectroscopy shows that the binding of the C14 and C16 alkyl trimethylammonium compounds to the mutant is similar to that of the corresponding fatty acids to the wild-type enzyme. Paramagnetic relaxation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 A closer to the iron, than seen in the wild-type, although this difference is much smaller ( approximately 0.2 A) in the ferrous state of the complex. The binding of a substrate having the same charge as residue 47 to the ferric state of the enzyme is roughly ten times weaker than that of a substrate having the opposite charge (and thus is able to make an ion-pair interaction with this residue). The results are discussed in the light of the three-dimensional structure of the enzyme.

| S-EPMC1218827 | biostudies-other

Dependence of micelle size and shape on detergent alkyl chain length and head group.

Project description:Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ?16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

| S-EPMC3648574 | biostudies-literature

Rugosibacter aromaticivorans gen. nov., sp. nov., a bacterium within the family Rhodocyclaceae, isolated from contaminated soil, capable of degrading aromatic compounds.

Project description:A bacterial strain designated Ca6T was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil from the site of a former manufactured gas plant in Charlotte, NC, USA, and linked phylogenetically to the family Rhodocyclaceae of the class Betaproteobacteria. Its 16S rRNA gene sequence was highly similar to globally distributed environmental sequences, including those previously designated 'Pyrene Group 1' demonstrated to grow on the PAHs phenanthrene and pyrene by stable-isotope probing. The most closely related described relative was Sulfuritalea hydrogenivorans strain sk43HT (93.6?% 16S rRNA gene sequence identity). In addition to a limited number of organic acids, Ca6T was capable of growth on the monoaromatic compounds benzene and toluene, and the azaarene carbazole, as sole sources of carbon and energy. Growth on the PAHs phenanthrene and pyrene was also confirmed. Optimal growth was observed aerobically under mesophilic temperature, neutral pH and low salinity conditions. Major fatty acids present included summed feature 3 (C16?:?1?7c or C16?:?1?6c) and C16?:?0. The DNA G+C content of the single chromosome was 55.14? mol% as determined by complete genome sequencing. Due to its distinct genetic and physiological properties, strain Ca6T is proposed as a member of a novel genus and species within the family Rhodocyclaceae, for which the name Rugosibacter aromaticivorans gen. nov., sp. nov. is proposed. The type strain of the species is Ca6T (=ATCC TSD-59T=DSM 103039T).

| S-EPMC5797942 | biostudies-literature

An improved procedure for the preparation of alkyl sulphate esters suitable for the study of secondary alkylsulphohydrolase enzymes.

Project description:A simple, rapid and convenient method for the synthesis of secondary alkyl sulphate esters is described in which the sodium alkoxide of the parent alcohol is sulphated by using triethylamine-SO3 complex. The procedure gives relatively good yields, even for the sulphation of long-chain alcohols and those in which the hydroxy group is remote from the terminal carbon atoms. Positional isomerization, arising from the migration of the hydroxy group along the carbon chain, is absent, and resolved enantiomers of alcohols react with complete retention of configuration.

| S-EPMC1162507 | biostudies-other

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