Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity.

Abstract

Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity. This region begins 275-310 amino acids from the amino terminus of each P-450, continues for =170 residues, and ends 35-50 amino acids before the carboxyl terminus. The region can be divided into four domains of sequence similarity, each possessing its own pattern of invariant, conserved, and variable amino acids. The four domains are 56,20, 59, and 28 residues long and are connected by three shorter segments of limited sequence similarity. The number of residues in these short segments varies with the P-450 protein but ranges from 0 to 20 residues. Consensus sequences based on these similarities can be used to determine whether the sequence of an unidentified peptide resembles that expected for a P-450. Sequence similarities between proteins sometimes reflect constraints imposed by the requirements of a common function. The fourth domain of the P-450s, for example, contains an invariant cysteine that provides the axial thiolate ligand to the heme iron. Other relationships between the four domains and P-450 function can be examined by in vitro mutagenic procedures that alter the conserved amino acids or modify the distance between domains.The P-450 cytochromes are components in monooxygenase systems that catalyze a wide variety of oxidative reactions in prokaryotes and eukaryotes. These reactions include steps in the synthesis and degradation of such compounds as cholesterol, steroid hormones, and prostaglandins. P-450 proteins are also involved in the metabolism of drugs and in the activation and inactivation of carcinogens. A recent analysis of >60 P-450s from eukaryotes and one prokaryote led to the organization of the known P-450s into 10 different families with a total of 15 subfamilies. Amino acid sequences within a P-450 family are >36% similar, while sequences within a subfamily are :70% similar (1). Although sequence relatedness between P-450 families is low, these families are still grouped into one P-450 superfamily according to standard criteria (2).The eukaryotic branch of the cytochrome P-450 superfamily arose >1000 million years ago (1, 3) and sequence similarities among these ancient families might identify conserved domains of structure or function. Since complete amino acid sequences had been published for proteins from eight eukaryotic P-450 families, we used a representative sequence from each family in a series of sequence comparisons. A multiple alignment of the eight sequences revealed one region of similarity shared by all. Sequence similarities in this region reside in four domains connected by short segments of variable length. The region will provide a focus for experiments that probe the relationship of amino acid sequence to structure and function in the P-450 superfamily. METHODS Amino Acid Sequences. The source for each eukaryotic P-450 sequence used in these comparisons was as follows: LAw, rat liver (4); c, rat liver (5); 17a, bovine adrenal cortex (6); scc,...

The Pea Gene LH Encodes ent-Kaurene Oxidase

“…The lh-1 and lh-3 putative proteins are full length and contain the substrate-binding and catalytic domains common to cytochrome P450 monooxygenases (Kalb and Loper, 1988; Fig. 4) and, therefore, may have some residual ent-kaurene oxidase activity even though this was not observed during expression of lh-1 in yeast (Table II).…”

“…4; Kalb and Loper, 1988) and, therefore, would be expected to be a null mutation. The PsKO1 sequence from the lh-3 mutant plants had a single-base change of guanine to adenine, which translates to a change from Val to Met in the putative protein (Fig.…”

“…The fungal P450-1 and P450-4 GA biosynthesis enzymes with ent-kaurene oxidase and ent-kaurenoic acid oxidase activity were also included. Kalb and Loper, 1988) are labeled, and cytochrome P450 highly conserved amino acids of known significance are underlined. The catalytic A domain is associated with substrate binding (Poulos et al, 1985) and oxygen pocket (Atkins and Sligar, 1988;Imai et al, 1989) and traverses the distal surface of the haem (Poulos et al, 1985).…”

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.)

“…The best values were 25% with animal LAH (CYP4A group) and 23% with several members of the CYP3A group (20). The similarity with other P-450 proteins was most pronounced in the four blocks of amino acids that are conserved in eight P-450 families (10) (Fig. 4).…”

“…It contained an EcoRI fragment of 1 kb and an open reading frame throughout the DNA sequence. A search in the SWISSPROT library showed that the deduced protein possessed significant similarity with the blocks A, B, and C, which are conserved in many P-450 families (10). The alignment with these proteins also predicted that pCrosl represented an EcoRI subfragment of a larger mRNA, and that the 5' and the 3' end of the mRNA were missing from this cDNA.…”