Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.

Abstract

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POXS genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the ,8-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains and POXS genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the "-oxidation pathway has been functionally blocked.Peroxisomes represent a class of organelles that are ubiquitous among eukaryotic organisms and are involved in a variety of metabolic processes such as ,8-oxidation and the degradation of hydrogen peroxide. The importance of these organelles to cellular metabolism has been made clear by the recent discovery of a new class of inherited peroxisomal disorders such as Zellweger syndrome, which has been reported to be due to a recessive mutation that abolishes the import of peroxisomal matrix proteins (11). However, the molecular events leading to compartmentalization in eukaryotic cells and to the localization of peroxisomal proteins are still largely unknown and are the subject of intense study.The yeast Candida tropicalis is an important organism for the study of peroxisome biogenesis and the localization of peroxisomal proteins, since these organelles are rapidly and abundantly induced following growth on either alkane or fatty acid substrates (10). At least 18 different peroxisomal proteins have been detected in oleic acid-induced cells, and several of these have been identified as enzymes associated with the 1-oxidation pathway (4). Localization of acyl coenzyme A (acyl-CoA) oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activities has shown that 13-oxidation takes place solely in the peroxisomes of this yeast (14). Peroxisomal acyl-CoA oxidase is an octomeric flavoprotein (molecular weight 600,000) which catalyzes the first reaction in the 1-oxidation pathway by the stoichiometric conversion of acyl-CoA to enoyl-CoA for substrates with chain lengths from 4 to 20 carbons (12). The genes encoding the subunits of distinct acyl-CoA oxidase isozymes (7-9), designated POX4 and POX5, have been cloned and sequenced. These genes encode polypeptides (designated PXP4 and PXP5) composed of 708 and 661 amino acids, respectively, following the * Corresponding author.removal of the initiating methionine during maturation (8). Since these polypeptides were found to share 63% homology, it was suggested that the two acyl-CoA oxidase isozymes arose by a gene duplication event followed by a localized exchange or replacement of functional domains. However, the functional differences between these isozymes, if any, have not been determined. A third member of the acyl-CoA oxidase gene family has been identified on the basis of sequence homology as P...

Biosynthesis of long-chain dicarboxylic acid monomers from renewable resources. Final technical report

“…In strain ATCC 20962, the genes coding for enzymes in the first step of fatty acid (3-oxidation have been disrupted so that the yeast can no longer use fatty acids as a 4-15 carbon source. In strain ATCC 20987, multiple copies of cytochrome P450 and reductase genes of the w-hydroxylase system have been introduced into the~-oxidation disrupted strain [2,3].…”

“…Then the electrostatic contribution to the activity coefficient is consistent with the Scatchard-Kirkwood equation, and the use of parameter K, is justified since it can be viewed as accounting for both salting-in (electrostic) and salting-out (hydrophobic) effects, the sum of which determines the volubility of the dia.cid at different salt concentrations [2,3].…”

“…It maybe possible to anticipate such an effect from the analysis of salt effects on hydrophobic interactions in precipitation (of proteins) by Melander and Horvath [2], who showed that:…”

“…The precipitation characteristics of the DCA were correlated with Cohn's equation, which has the form [2]: This correlation was used to fit the data since the long chain dicarboxylic acids can be represented as two charged spheres of raclius a, with a large distance, d, between the spheres such that ald <<1. Then the electrostatic contribution to the activity coefficient is consistent with the Scatchard-Kirkwood equation, and the use of parameter K, is justified since it can be viewed as accounting for both salting-in (electrostic) and salting-out (hydrophobic) effects, the sum of which determines the volubility of the dia.cid at different salt concentrations [2,3].…”

“…For example, azelaic (C9) and sebacic (C 10) acids are used to make aliphatic polyesters which, in turn, are used in fibers, films, casting resins, plasticizers, synthetic lubricants, and adhesives [1,2]. Brassylic acid (C13) is used in the manufacture of synthetic musk as well as nylon 13,13.…”