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

Brock, Barry J.; Waterman, Michael R.

doi:10.1006/abbi.1999.1557

Cited by 18 publications

(4 citation statements)

References 38 publications

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“…Small sets of chimeric P450s have been constructed previously for investigations of sequence-structure-function relationships [ 38, 39]. The MSA of natural protein families are also widely used for this purpose.…”

Section: Resultsmentioning

confidence: 99%

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

et al. 2006

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Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ˜3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.

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Section: Resultsmentioning

confidence: 99%

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

et al. 2006

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“…For example, a chimeric heme domain significantly more thermostable than any of the parents was identified (T 50 = 62°C versus 55°C for the most stable parent) [9]; subsequent analysis of more than 200 chimeric heme domains identified many thermostable proteins [Yougen Li et al unpublished data]. Our previous study of selected chimeras of the A1 and A2 heme domains showed that chimeragenesis could also generate activities not exhibited by the parents [10], as has also been reported for recombination of mammalian P450s [11,12].…”

Section: Introductionmentioning

confidence: 62%

Diversification of Catalytic Function in a Synthetic Family of Chimeric Cytochrome P450s

Landwehr

Carbone

Otey

et al. 2007

Chemistry & Biology

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We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a "surrogate" for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping.

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“…As shown here, both the porcine and bovine enzymes have an isoleucine in this position, even though porcine retains its C17,20-lyase activity with 17α-OH P4 while bovine appears deficient in this activity. Studies examining the activities of chimeric rat/human enzymes also led Brock and Waterman (2000) to conclude that phenylalanine 343 was important to the lyase activity of rat only in the context of the rat protein. Similarly, Gilep and colleagues studied 10 different chimeras made between bovine and guinea pig.…”

Section: A-hydroxylase/c1720-lyase Activitiesmentioning

confidence: 96%

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

Shet

Fisher

Tremblay

et al. 2007

Drug Metabolism Reviews

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The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17 alpha-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17 alpha-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17 alpha-hydroxylated P4 and P5 (17 alpha-OH P4 and 17 alpha-OH P5) followed by the C17,20-lyase reaction for the conversion of these C(21)-17 alpha-hydroxylated steroids to C(19)-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b(5) (b(5))-stimulated C17,20-lyase activity that converts 17 alpha OH-P4 to androstenedione (AD) but also converts 17 alpha-OHP5 to dehydroepiandrosterone (DHEA); (b) guinea pig P450c17 possesses a b(5)-stimulated C17,20-lyase activity that converts 17 alpha-OH P4 to AD but does not convert 17 alpha-OH P5 to DHEA., and (c) bovine P450c17 possesses a b(5)-stimulated C17,20-lyase activity that converts 17 alpha-OH P5 to DHEA but does not convert 17 alpha-OH P4 to AD. Thus, the P450c17 of each species differs in its ability to catalyze in vitro the conversion of C(21)-steroids to C(19)-steroids. In addition, each P450c17 is capable of catalyzing additional hydroxylation reactions leading to low levels of 2 alpha-, 6 beta-, 16- and 21-hydroxy-metabolites. Porcine P450c17 also catalyzes the b(5)-dependent synthesis of andien-beta (androsta-5,16-dien-3beta-ol) from P5. When the amino acid sequences of the three P450c17s were aligned there was an approximate 50% variation in the alignment identity (227 differences in the sequences of 509 amino acids). Alignment did not permit the assignment of specific amino acids or domains to the observed differences in enzymatic activities.

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

Cited by 18 publications

References 38 publications

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Diversification of Catalytic Function in a Synthetic Family of Chimeric Cytochrome P450s

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

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