Search

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. OCIS codes: 140.0140, 140.3425, 120.0120, 120.3930, 120.3940, 300.6190.

Research has been conducted on arylketone monooxygenase purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. Results indicate that this monooxygenase has characteristics of the Baeyer-Villiger-type monooxygenase.

Stable tropic acid dehydrogenase from Pseudomonas sp. strain AT3 was purified by ion-exchange chromatography and analyzed to determine the enzymology of microbial phenyl acetic acid oxidation. Analysis of purified Pseudomonas sp. tropic acid dehydrogenase indicated the presence of nicotinamide adenine dinucleotide (NAD+) linkage. The NAD+-linked isocitrate and malate dehydrogenases also mediated the conversion of tropic acid to phenylacetic acid without forming a phenylmalonic semialdehyde intermediate.

Pseudomonas sp. strain AT3 hydrolyzes atropine to form nortropine, and then 6-hydroxycyclohepta-1,4-dione. The beta-diketone 4,6-dioxoheptanoate cleavage point produces either succinate and acetone or 4-oxopentanoate and acetate. Nitrogen elimination produces 6-hydroxycyclohepta-1,4-dione. The oxidation of 6-hydroxycyclohepta-1,4-dione by NAD+ -specific dehydrogenase produces cyclohepta-1,3,5-trione. The AT3 strain fails to grow with acetone and the compound evaporates into the atmosphere.

Growth of Aspergillus fumigatus ATCC 28282 occurs on 4-ethylphenol through a metabolic pathway and involves hydroxylation and oxidation. Hydroxylation is stimulated by a NADPH-dependent enzyme. A fumigatus extract grown on succinate medium does not perform any enzyme activity.

The utilization of p-cresol as the sole source of carbon and energy for Aspergillus fumigatus is discussed. Cell extracts from the fungi were subjected to thin-layer chromatography, gas-chromatography-mass spectrometry, monitoring of oxygen consumptions and enzyme and protein assays. The growth of A. fumigatus on p-cresol was demonstrated as two routes were determined converging at the protachetuic acid.

The authors have investigated periplasmic cytochrome c from the lupanine-utilizing Pseudomonas putida strain. The aim of the study has been the cloning, sequencing and heterologously expressing this cytochrome, and the determination of the cytochrome's molecular size via the use of electrospray mass spectrometry.

Research has been conducted on 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1. Results demonstrate that this compound is affected by dehydrogenation of its substrate and produces quinone methide which is then hydrated to alcohol.

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 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles. [ABSTRACT FROM AUTHOR]

4-Hydroxyisophthalate hydroxylase has been purified from cells grown with 2,4-xylenol and gave a single protein band on polyacrylamide-gel electrophoresis after a 24-fold purification. In the reaction, 4-hydroxyisophthalate was converted to protocatechuate by an oxidative decarboxylation and the reaction stoichiometry is typical of a monooxygenase with an external electron donor. The enzyme can function with either NADH (Km 105 μM) or NADPH (Km 71 μM). Besides 4-hydroxyisophthalate (Km 42 μM), 5-sulphosalicylate was the only aromatic substrate found for the enzyme. The enzyme has a molecular weight of about 103000, consisting of two identical polypeptide chains, and contains one mole of FAD per mole of protein. The FAD could not be replaced by FMN m reconstituting active enzyme from native apoenzyme. The formation of an enzyme-substrate complex was detected by the change in flavin spectrum when 4-hydroxyisophthalate was added to the enzyme. Rapid reduction of the enzyme-bound FAD by NADPH only occurred in the presence of substrate. No stable flavin semiquinones were observed in the titration with NADPH or during anaerobic photoreduction in the presence of EDTA. Although a number of substrate analogues inhibit the enzyme no non-hydroxylatable effectors of the NADPH oxidation were found. The enzyme was inhibited by sulphydryl-binding reagents and not protected by substrate. Addition of sulphydryl compounds was essential for stabilization of the enzyme during purification. Extracts of cells grown with 2,4-xylenol and 4-hydroxyisophthalate also contain a separate 4-hydroxybenzoate hydroxylase. [ABSTRACT FROM AUTHOR]