Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. 2000

R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242, USA. rebecca-parales@uiowa.edu

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

UI MeSH Term Description Entries
D007203 Indigo Carmine Indolesulfonic acid used as a dye in renal function testing for the detection of nitrates and chlorates, and in the testing of milk. Indigotindisulfonate Sodium,Soluble Indigo Blue,(delta-2,2'-biindole)-3,3'-dione,2-(1,3-Dihydro-3-oxo-5-sulpho-2H-indol-2-ylidene)-3- oxoindoline-5-sulphonic acid,D&C Blue NO. 6,FD&C Blue No. 2,Indigo,Indigo Blue,Indigo Disulfonate,Indigotin,Indigotindisulfonate,Indigotindisulfonic Acid,Carmine, Indigo,Indigo Blue, Soluble
D007211 Indoles Benzopyrroles with the nitrogen at the number one carbon adjacent to the benzyl portion, in contrast to ISOINDOLES which have the nitrogen away from the six-membered ring.
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D009097 Multienzyme Complexes Systems of enzymes which function sequentially by catalyzing consecutive reactions linked by common metabolic intermediates. They may involve simply a transfer of water molecules or hydrogen atoms and may be associated with large supramolecular structures such as MITOCHONDRIA or RIBOSOMES. Complexes, Multienzyme
D009281 Naphthalenes Two-ring crystalline hydrocarbons isolated from coal tar. They are used as intermediates in chemical synthesis, as insect repellents, fungicides, lubricants, preservatives, and, formerly, as topical antiseptics.
D010105 Oxygenases Oxidases that specifically introduce DIOXYGEN-derived oxygen atoms into a variety of organic molecules. Oxygenase
D010616 Phenanthrenes POLYCYCLIC AROMATIC HYDROCARBONS composed of three fused BENZENE rings.
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D003470 Culture Media Any liquid or solid preparation made specifically for the growth, storage, or transport of microorganisms or other types of cells. The variety of media that exist allow for the culturing of specific microorganisms and cell types, such as differential media, selective media, test media, and defined media. Solid media consist of liquid media that have been solidified with an agent such as AGAR or GELATIN. Media, Culture
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli

Related Publications

R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
June 2006, Applied microbiology and biotechnology,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
March 2006, Applied and environmental microbiology,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
May 2003, Journal of industrial microbiology & biotechnology,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
August 2001, Journal of industrial microbiology & biotechnology,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
October 2003, The Journal of biological chemistry,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
May 1998, Journal of bacteriology,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
April 2010, FEBS letters,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
April 2003, Proceedings of the National Academy of Sciences of the United States of America,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
August 2006, Journal of the American Chemical Society,
R E Parales, and K Lee, and S M Resnick, and H Jiang, and D J Lessner, and D T Gibson
January 1990, Biochemical and biophysical research communications,
Copied contents to your clipboard!