Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

<p>Abstract</p> <p>Background</p> <p>β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured <it>in vitro </it>using His-tag purified proteins and <it>in vivo </it>in a β-carotene-accumulating <it>E. coli </it>system.</p> <p>Results</p> <p>Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our <it>ab initio </it>model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.</p> <p>Conclusions</p> <p>Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.</p

Achromobacter xylosoxidans as a new microorganism strain colonizing high-density polyethylene as a key step to its biodegradation

This study presents results of research on isolation new bacteria strain Achromobacter xylosoxidans able to effect on the structure of high-density polyethylene (HDPE), polymer resistant to degradation in environment. New strain of A. xylosoxidans PE-1 was isolated from the soil and identified by analysis of the 16S ribosome subunit coding sequences. The substance to be degraded was HDPE in the form of thin foil films. The foil samples were analyzed with Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) as well as scanning electron microscope (SEM), and the results revealed degradation of chemical structure of HDPE. About 9 % loss of weight was also detected as a result of A. xylosoxidans PE-1 effect on HDPE foil. On the basis of comparative spectral analysis of the raw material before the bacteria treatment and the spectrum from a spectra database, it was assumed that the HDPE was the only source of carbon and energy for the microorganisms. No fillers or other additives used in the plastic processing were observed in HDPE before experiments. This is the first communication showing that A. xylosoxidans is able to modify chemical structure of HDPE, what was observed both on FTIR, in mass reduction of HDPE and SEM analysis. We also observed quite good growth of the bacteria also when the HDPE was the sole carbon source in the medium. These results prove that A. xylosoxidans is an organism worth applying in future HDPE biodegradation studies

Violaxanthin de-epoxidase, the xanthophyll cycle enzyme, requires lipid inverted hexagonal structures for its activity

Bilayer-forming lipids were shown to be ineffective in sustaining the enzymatic activity of violaxanthin de-epoxidase. On the other hand, non-bilayer-forming lipids, regard-less of their different chemical character, ensured high activity of violaxanthin de-epoxidase, resulting in conversion of violaxanthin to zeaxanthin. Our data indicates that the presence of lipids forming reversed hexagonal structures is necessary for violaxanthin de-epoxidase activity and this activity is dependent on the degree of unsaturation of the fatty acids. The significance of the reversed hexagonal phase domains in the conversion of violaxanthin into zeaxanthin in model systems and in the native thylakoid membranes is discussed