Affordable Access

Publisher Website

The Use of Random Chimeragenesis to Study Structure/Function Properties of Rat and Human P450c17

Authors
Journal
Archives of Biochemistry and Biophysics
0003-9861
Publisher
Elsevier
Publication Date
Volume
373
Issue
2
Identifiers
DOI: 10.1006/abbi.1999.1557
Keywords
  • P450
  • 17α-Hydroxlase
  • Steroidogenesis
  • Structure/Function
  • Random Chimeragenesis
Disciplines
  • Biology
  • Chemistry
  • Medicine

Abstract

Abstract The microsomal 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes the 17α-hydroxylase reaction required to produce cortisol, the major glucocorticoid in many species and the 17,20-lyase activity required for the production of androgens in all species. Utilizing the technique of random chimeragenesis we have attempted to map regions of primary sequence that contribute to the species-specific biochemical differences between rat and human P450c17. We have previously reported significant differences between rat and human P450c17 in their activities, stability and substrate-dependent coupling efficiencies even though they share 68% amino acid identity. Identification of the regions of primary sequence that contribute to each of these properties would be helpful in understanding the structure/function relationships in this enzyme. A single plasmid containing the cDNAs encoding both enzymes in a tandem orientation was constructed. This plasmid was linearized at unique restriction sites and used to transform Escherichia coli. A three-step screening protocol identified five chimeras with a uniform distribution of 5′ rat and 3′ human sequence. All chimeric proteins yield the characteristic reduced-CO difference spectra, indicating proper folding. The chimeras exhibit a range of stability and activities that are not consistent with the degree of parental primary sequence. A chimera containing 301 N-terminal rat P450c17 amino acids and lacking the rat P450c17 phenylalanine 343, had the highest lyase activity. Generation of these functional rat/human chimeras suggests that the tertiary structures of rat and human P450c17 are sufficiently conserved to allow proper folding of chimeric enzymes. However, the properties of these chimeras did not permit identification of a region of primary sequence that contributes to a species-specific property of rat and human P450c17. Stability of these chimeras and insight into the presence of secondary structural elements is discussed.

There are no comments yet on this publication. Be the first to share your thoughts.

Statistics

Seen <100 times
0 Comments

More articles like this

The use of random chimeragenesis to study structur...

on Archives of Biochemistry and B... Jan 15, 2000

Studies of distant members of the P450 superfamily...

on Archives of Biochemistry and B... Oct 15, 1996

Studies of Distant Members of the P450 Superfamily...

on Archives of Biochemistry and B... Jan 01, 1996

Expression and purification of functional human 17...

on Journal of Biological Chemistr... Sep 15, 1993
More articles like this..