Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins.

T R Sutter,D Sanglard,J C Loper,D Sangard

doi:10.1016/s0021-9258(17)46240-5

Abstract

The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis lambda gt11 expression library and colony hybridization of a C. tropicalis genomic library. The C. tropicalis CPR gene produces a 2.35-kilobase mRNA transcript, levels of which were shown to be increased 16-fold in cells grown on tetradecane relative to cells grown on glucose as the sole carbon source. A 3-kilobase DNA fragment was sequenced, including 554 and 397 base pairs of 5'- and 3'-noncoding sequence, respectively. A single open reading frame of 2040 base pairs was identified and predicts a 76,683-Da polypeptide of 680 amino acid residues. The deduced C. tropicalis CPR amino acid sequence was compared with each of the CPR sequences reported from other organisms and invariant residues were identified. Multiple pairwise alignments of divergent members of protein families, previously recognized for their sequence similarities in their respective binding domains for FMN, FAD, and NADPH, have allowed identification of a subset of these invariant residues. From these analyses we infer the importance of 25 of the 680 amino acid residues.

Highlights

P-460 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis Xgtll expression library and colony hybridization of a C
The C. tropicalis CPR gene produces a 2.35-kilobase mRNA transcript, levels of which were shown to be increased 16-fold in cells grown on tetradecane relative to cells grown on glucose as the sole carbon source
Gene Isolation-Immunoscreening of a C. tropicalis hgtll expression library with polyclonal antiserum raised against the purified C. tropicalis CPR protein yielded eight positive clones, identified as clones 1, 2, 4, 6, 7, 9, 12, and 13

Summary

Introduction

P-460 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis Xgtll expression library and colony hybridization of a C. A single open reading frame of 2040 base pairs was identified and predicts a 76,683-Da polypeptide of 680 amino acid residues. Multiple pairwise alignments of divergent members of protein families, previously recognized for their sequence similarities in their respective binding domains for FMN, FAD, and NADPH, have allowed identification of a subset of these invariant residues. From these analyses we infer the importance of 25 of the 680 amino acid residues. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked “aduertisement”

Methods

Results

Conclusion