In vitro studies on the degradation of common rubber waste material with the latex clearing protein (Lcp1VH2) of Gordonia polyisoprenivorans VH2.

The enzymatic degradation of the rubber polymer poly(cis-1,4-isoprene), e.g. by the latex clearing protein Lcp1VH2 of Gordonia polyisoprenivorans VH2 has been demonstrated with latex milk or pure isoprene-rubber particles, recently. Unfortunately, carbon black filled vulcanized rubber (CFVR) making the biggest part of worldwide rubber wastes, contains several harmful additives making microbial and enzymatic rubber degradation challenging. However, this study demonstrates the successful enzymatic cleavage of industrially produced CFVR. The formation of the cleavage products, oligo(cis-1,4-isoprenoids), from incubating CFVR particles with Lcp1VH2 was detected by HPLC-MS. Various organic solvents were tested to remove harmful or inhibiting additives like antioxidants to enhance product formation. The pretreatment of CFVR particles, especially with chloroform or cyclohexane, significantly improved the degradation. It was also demonstrated that reducing the particles size and thus increasing the enzymatically accessible surface area of the particles led to a strong acceleration of the degradation process. Furthermore, ATR-IR analyses showed that Lcp1VH2 led to the functionalization of the rubber particle surface with carbonyl groups by cleaving isoprene chains, still linked to the particle. Both, the oligo(cis-1,4-isoprenoids) as well as the functionalized rubber particles, are potentially important products, which can be reused as fine chemicals or as additives in rubber production. The present study, showing the enzymatic degradation of common CFVR for the first time, takes an important step towards a new way of rubber waste disposal and indicates the economic feasibility of an efficient and environmentally friendly recycling process by using the rubber oxygenase Lcp1VH2.

Modification of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one and other secondary metabolites by methyltransferases from mycobacteria.

The opportunistic pathogen Pseudomonas aeruginosa, one of the most prevalent species in infections of the cystic fibrosis lung, produces a range of secondary metabolites, among them the respiratory toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (2-heptyl-4-hydroxyquinoline N-oxide, HQNO). Cultures of the emerging cystic fibrosis pathogen Mycobacteroides abscessus detoxify HQNO by methylating the N-hydroxy moiety. In this study, the class I methyltransferase MAB_2834c and its orthologue from Mycobacterium tuberculosis, Rv0560c, were identified as HQNO O-methyltransferases. The P. aeruginosa exoproducts 4-hydroxyquinolin-2(1H)-one (DHQ), 2-heptylquinolin-4(1H)-one (HHQ), and 2-heptyl-3-hydroxyquinolin-4(1H)-one (the 'Pseudomonas quinolone signal', PQS), some structurally related (iso)quinolones, and the flavonol quercetin were also methylated; however, HQNO was by far the preferred substrate. Both enzymes converted a benzimidazole[1,2-a]pyridine-4-carbonitrile-based compound, representing the scaffold of antimycobacterial substances, to an N-methylated derivative. We suggest that these promiscuous methyltransferases, newly termed as heterocyclic toxin methyltransferases (Htm), are involved in cellular response to chemical stress and possibly contribute to resistance of mycobacteria toward antimicrobial natural compounds as well as drugs. Thus, synthetic antimycobacterial agents may be designed to be unamenable to methyl transfer. ENZYMES: S-adenosyl-l-methionine:2-heptyl-1-hydroxyquinolin-4(1H)-one O-methyl-transferase, EC 2.1.1.

Pseudomonas Quinolone Signal molecule PQS behaves like a B Class inhibitor at the IQ site of mitochondrial complex I.

Pseudomonas aeruginosa is a Gram-negative bacterium of the proteobacteria class, and one of the most common causes of nosocomial infections. For example, it causes chronic pneumonia in cystic fibrosis patients. Patient sputum contains 2-heptyl-4-hydroxyquinoline N-oxide [HQNO] and Pseudomonas quorum sensing molecules such as the Pseudomonas quinolone signal [PQS]. It is known that HQNO inhibits the enzyme activity of mitochondrial and bacterial complex III at the Qi (quinone reduction) site, but the target of PQS is not known. In this work we have shown that PQS has a negative effect on mitochondrial respiration in HeLa and A549 cells. It specifically inhibits the complex I of the respiratory chain. In vitro analyses showed a partially competitive inhibition with respect to ubiquinone at the IQ site. In competing studies with Rotenone, PQS suppressed the ROS-promoting effect of Rotenone, which is typical for a B-type inhibitor. Prolonged incubation with PQS also had an effect on the activity of complex III.

High yield production of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed batch fermentations using a recombinant strain of Escherichia coli.

The enzymatic degradation of rubber with the latex clearing protein (Lcp1VH2) from Gordonia polyisoprenivorans VH2, is a promising option as an environmentally friendly and economical solution to treat the enormous amount of rubber waste. Here we present a fed batch fermentation process on a 10 L scale, using E.coli C41 pET23a(+)::Hislcp1VH2 and a modified defined mineral salt medium, designed for high cell densities, for a proper synthesis of Lcp1VH2. Particularly, providing complex media components, as well as hemin, as precursor of the essential heme b cofactor, resulted in a 2.9-fold higher yield of active Lcp1VH2 with increased specific activity, due to a better occupancy of the enzyme with the cofactor. Based on this optimization, the fed batch fermentation with an initial glucose feed, followed by a lactose-glycerol feed, finally gained a cell dry weight of 60 g L-1 and a yield of 223 mg L-1 of soluble, active Lcp1VH2. Compared to a recently published fermentation process, which used a complex auto-induction medium, we significantly increased the biomass up to nearly 10-fold and the total Lcp1VH2 yield up to 3.7-fold. Thereby we reduced the costs for the medium by 75 %, taking the next step towards industrial production of rubber degrading enzymes.

Efficient modification of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4-one by three Bacillus glycosyltransferases with broad substrate ranges.

Glycosylation of natural and synthetic products can alter the physical, chemical and pharmacological properties of the aglycon. Conversion of 2-heptyl-1-hydroxyquinolin-4-one (HQNO), a potent respiratory inhibitor produced by Pseudomonas aeruginosa, to the less toxic 2-heptyl-1-(ß-D-glucopyranosydyl)-quinolin-4-one, was recently demonstrated for Bacillus subtilis strain 168. In this study, we compared the genomes of several Bacillus spp. to identify candidate enzymes for HQNO glucosylation. All three (putative) UDP-glycosyltransferases (GT) of B. subtilis 168 tested, YjiC, YdhE and YojK, were capable of HQNO glucosylation, with YjiC showing the highest turnover rate (kcat) of 4.6 s-1, and YdhE exhibiting the lowest Km value for HQNO of 9.1 µM. All three GT predominantly utilized UDP-glucose, but YdhE was similarly active with TDP-glucose. Among the aglycons tested, HQNO was the preferred substrate of all three GT, but they also showed activities toward the P. aeruginosa exoproducts pyocyanin, 2-heptyl-3-hydroxyquinolin-4(1H)-one (the Pseudomonas quinolone signal) and 2,4-dihydroxyquinoline, the plant derived antimicrobials vanillin and quercetin, and the macrolide antibiotic tylosin A. Our results underline the promiscuity and substrate flexibility of YjiC, YdhE and YojK, and suggest a physiological role in natural toxin resistance of B. subtilis. Especially YdhE appears to be an attractive biocatalyst for the glycoengineering of natural products.

Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS.

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine-metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.

A Novel Steroid-Coenzyme A Ligase from Novosphingobium sp. Strain Chol11 Is Essential for an Alternative Degradation Pathway for Bile Salts.

Bile salts such as cholate are steroid compounds with a C5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7α-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-seco degradation pathway used by other steroid-degrading bacteria indicating that strain Chol11 uses an alternative pathway. A reciprocal BLASTp analysis showed that known side chain degradation genes from other cholate-degrading bacteria (Pseudomonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1) were not found in the genome of strain Chol11. The characterization of a transposon mutant of strain Chol11 showing altered growth with cholate identified a novel steroid-24-oyl-coenzyme A ligase named SclA. The unmarked deletion of sclA resulted in a strong growth rate decrease with cholate, while growth with steroids with C3 side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-Δ4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid (DOCDA), were shown to be likely physiological substrates of SclA. Furthermore, a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with an additional double bond in the side chain was identified. These results support the hypothesis that Novosphingobium sp. strain Chol11 harbors an alternative pathway for cholate degradation, in which side chain degradation is initiated by the CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknown enzymes different from those of other steroid-degrading bacteria.IMPORTANCE This study provides further evidence of the diversity of metabolic pathways for the degradation of steroid compounds in environmental bacteria. The knowledge about these pathways contributes to the understanding of the CO2-releasing part of the global C cycle. Furthermore, it is useful for investigating the fate of pharmaceutical steroids in the environment, some of which may act as endocrine disruptors.

Chemical Modification and Detoxification of the Pseudomonas aeruginosa Toxin 2-Heptyl-4-hydroxyquinoline N-Oxide by Environmental and Pathogenic Bacteria.

2-Heptyl-4-hydroxyquinoline N-oxide (HQNO), a major secondary metabolite and virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa, acts as a potent inhibitor of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this study, we demonstrate the previously unknown capability of environmental and pathogenic bacteria to transform and detoxify this compound. Strains of Arthrobacter and Rhodococcus spp. as well as Staphylococcus aureus introduced a hydroxyl group at C-3 of HQNO, whereas Mycobacterium abscessus, M. fortuitum, and M. smegmatis performed an O-methylation, forming 2-heptyl-1-methoxy-4-oxoquinoline as the initial metabolite. Bacillus spp. produced the glycosylated derivative 2-heptyl-1-(ß-d-glucopyranosydyl)-4-oxoquinoline. Assaying the effects of these metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed that their EC50 values were up to 2 orders of magnitude higher than that of HQNO. Furthermore, cellular levels of reactive oxygen species were significantly lower in the presence of the metabolites than under the influence of HQNO. Therefore, the capacity to transform HQNO should lead to a competitive advantage against P. aeruginosa. Our findings contribute new insight into the metabolic diversity of bacteria and add another layer of complexity to the metabolic interactions which likely contribute to shaping polymicrobial communities comprising P. aeruginosa.

Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies.

BACKGROUND: Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni(2+) and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant. RESULTS: Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni(2+) was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni(2+) ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity. CONCLUSIONS: Heterologous formation of catalytically active, native QueD holoenzyme requires Ni(2+), Co(2+) or Mn(2+), i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni(2+) most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.

Substrate-assisted O2 activation in a cofactor-independent dioxygenase.

In contrast to the majority of O2-activating enzymes, which depend on an organic cofactor or a metal ion for catalysis, a particular group of structurally unrelated oxygenases is functional without any cofactor. In this study, we characterized the mechanism of O2 activation in the reaction pathway of a cofactor-independent dioxygenase with an α/ß-hydrolase fold, which catalyzes the oxygenolytic cleavage of 2-alkyl-3-hydroxy-4(1H)-quinolones. Chemical analysis and electron paramagnetic resonance spectroscopic data revealed that O2 activation in the enzyme's active site is substrate-assisted, relying on single electron transfer from the bound substrate anion to O2 to form a radical pair, which recombines to a C2-peroxide intermediate. Thus, an oxygenase can function without a cofactor, if the organic substrate itself, after activation to a (carb)anion by an active-site base, is intrinsically reactive toward molecular oxygen.

Hydrolase-like properties of a cofactor-independent dioxygenase.

Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/ß-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/ß-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.