Genome Sequence Analysis of Two Pseudomonas putida Strains to Identify a 17-Hydroxylase Putatively Involved in Sparteine Degradation.

Two strains of Pseudomonas putida, Psp-LUP and Psp-SPAR, capable of growth on the quinolizidine alkaloids, lupanine and sparteine respectively, were studied here. We report the isolation of Psp-SPAR and the complete genome sequencing of both bacteria. Both were confirmed to belong to P.

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein from Stenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank.

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.

Optimizing modulation transfer spectroscopy signals for frequency locking in the presence of depleted saturating fields.

A theoretical model of modulation transfer spectroscopy (MTS) that includes pump beam depletion is presented and experimentally verified with data covering visible iodine transitions at 532, 543, and 612 nm. This model is used to determine the values for pressure, interaction length, and saturation intensity that yield maximum MTS signals for frequency locking to iodine transitions. The approach is demonstrated for iodine transitions at 532, 633, and 778 nm, with the results showing that theoretically the frequency instability scales inversely to the absorption coefficient.

Export of a heterologous cytochrome P450 (CYP105D1) in Escherichia coli is associated with periplasmic accumulation of uroporphyrin.

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli.

A cytochrome c from a lupanine-transforming Pseudomonas putida strain is expressed in Escherichia coli during aerobic cultivation and efficiently exported and assembled in the periplasm.

We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain. Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm ( approximately 4 mg of hemoprotein/liter of culture). The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine.

Alkylphenol biotransformations catalyzed by 4-ethylphenol methylenehydroxylase.

4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product.

The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida.

Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida. It dehydrogenates the substrate, which can then be hydrated. It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ.

Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp.

The gene encoding the enzyme lupanine hydroxylase was isolated by PCR using chromosomal DNA from a lupanine-utilizing Pseudomonas sp. as template and primers based on the sequences of the N- and C-termini of the purified protein. The derived sequence for the mature gene product gave a protein with an M (r) of 72256, in good agreement with the value found by SDS/PAGE of the pure enzyme, and contained the sequences of several peptides obtained after endoproteinase Lys-C digestion of the pure enzyme.