The Copper-Enzyme Family of Dopamine β-Monooxygenase and Peptidylglycine α-Hydroxylating Monooxygenase: Resolving the Chemical Pathway for Substrate Hydroxylation
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Dopamine β-monooxygenase (DβM)2 and peptidylglycine α-hydroxylating monooxygenase (PHM) belong to a small class of copper proteins found exclusively in higher eukaryotes. These physiologically important enzymes catalyze the transformation of dopamine to norepinephrine (DβM) (Equation 1) and C-terminal glycine-extended peptides to their α-hydroxylated products (PHM) (Equation 2). Although their substrate specificities are grossly different, these enzymes greatly resemble each other in many other respects.Keywords:
Hydroxylation
Dopamine β-monooxygenase (DβM)2 and peptidylglycine α-hydroxylating monooxygenase (PHM) belong to a small class of copper proteins found exclusively in higher eukaryotes. These physiologically important enzymes catalyze the transformation of dopamine to norepinephrine (DβM) (Equation 1) and C-terminal glycine-extended peptides to their α-hydroxylated products (PHM) (Equation 2). Although their substrate specificities are grossly different, these enzymes greatly resemble each other in many other respects.
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Abstract CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
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Filamentous fungi are capable of catalyzing regio- and stereoselective hydroxylation on an extensive array of natural and synthetic hydrophobic organic substrates. A significant benefit of fungal hydroxylation is that non-activated carbon centers can be functionalized in ways that may not be easily emulated by classical organic means. The enzymes thought to perform the hydroxylations are cytochrome P450 monooxygenases. This review presents the evidence for the role of cytochrome P450s and summarizes the broad spectrum of substrates hydroxylated by various filamentous fungi in the twenty years prior to August 2000. Whole cell systems are generally preferred because monooxygenases that catalyze these reactions have not been isolated and characterized. Optimization of the hydroxylation conditions and the ability to accurately predict the biotransformation product(s) are necessary future developments before widespread synthetic practicality is achieved. Several recent developments in optimization techniques are also discussed including the use of protecting groups, varied experimental conditions, and the use of additives to the whole cell systems.
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Abstract CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
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We describe the synthesis and reactivity of new mononuclear copper(I) complexes 4a–c, which can be considered to be copper monooxygenase models and can be used to achieve hydroxylation of aliphatic C–H bonds by dioxygen activation.
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CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
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Abstract Steroids are the second largest class of drugs with a wide range of pharmacological properties. Hydroxylation of steroids seriously affects their biological activities and other properties. However, steroids are mostly sp 3 hybridized carbons with numerous C−H bonds far from the functional group that can activate them, and achieving regio‐ and stereo‐selective hydroxylation on steroids is a highly challenging task that is almost impossible to achieve using modern organic synthesis techniques. Interestingly, cytochrome P450 monooxygenases possess the ability to catalyse regio‐ and stereo‐selective oxidations of nonactivated C−H bonds in complex organic molecules under mild conditions. This review summarizes the P450s identified and engineered in recent years that can catalyse steroid nucleus hydroxylation stereo‐ and regio‐selectively. magnified image
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