Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes

JBC Papers in Press. Published on December 6, 2017 as Manuscript RA117.000504

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000504

P450 17A structure and functionꢀ

S

Inherent steroid 17a,20-lyase activity in defunct cytochrome P450 17A enzymes

‡

,1,2

‡,1,3

§

‡

‡,4

Eric Gonzalez

, Kevin M. Johnson

‡

, Pradeep S. Pallan , Thanh T. N. Phan , Wei Zhang

,

||

‡

Li Lei , Zdzislaw Wawrzak , Francis K. Yoshimoto , Martin Egli , and

‡

,5

F. Peter Guengerich

‡

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee

§

3

7232-0146, Life Sciences Collaborative Access Team, Sector 21, Advanced Photon Source, Argonne

|

National Laboratory, Argonne, Illinois 60439, and Department of Chemistry, University of Texas at San

Antonio, San Antonio, Texas 78249

Running title: P450 17A structure and function

Address correspondence to: Prof. F. Peter Guengerich, Department of Biochemistry, Vanderbilt

University School of Medicine, 638B Robinson Research Building, 2200 Pierce Avenue, Nashville,

Tennessee 37232-0146, Telephone:

f.guengerich@vanderbilt.edu

1

(615) 322-2261; FAX:

1

(615) 343-0704; E-mail:

Keywords: cytochrome P450, enzyme kinetics, x-ray crystallography, steroidogenesis, site-directed

mutagenesis, biomimetic models

ABSTRACT

Cytochrome P450 (P450) 17A1 catalyzes

the oxidations of progesterone and pregnenolone

and is the major source of androgens. The enzyme

catalytically

competent

kinetics),

plus

hydroxylated 17a-hydroxy steroids. The 17a-

3

+

OOH results indicate that a “Compound I” (FeO )

intermediate is capable of formation and can be

used to rationalize the products. We conclude that

zebrafish P450 17A2 is capable of lyase activity

with the 17a-OOH steroids because it can achieve

an appropriate conformation for lyase catalysis in

this system that is precluded in the conventional

reaction.

catalyzes both 17a-hydroxylation and

a

subsequent 17a, 20-lyase reaction, and several

mechanisms have been proposed for the latter step.

Zebrafish P450 17A2 catalyzes only the 17a-

hydroxylations. We previously reported high

similarity of the crystal structures of zebrafish

P450 17A1 and 17A2 and human P450 17A1

(

3

Pallan et al. (2015) J. Biol. Chem. 290, 3248-

268). Five residues near the heme, which

The human genome contains 57 genes

coding for cytochrome P450 (P450, CYP)

enzymes. These are involved in the metabolism of

a great variety of chemicals (1), including drugs,

chemical carcinogens, fatty acids, eicosanoids, and

fat-soluble vitamins (2). P450s also dominate the

metabolism of steroids (2, 3), with P450s 11A1,

17A1, 21A2, 11B1, 11B2, and 19A1 plus four

dehydrogenases accounting for most of the steps

between cholesterol and the androgens, estrogens,

glucocorticoids, and mineralocorticoids (3). P450

differed, were changed. We also crystallized this

five-residue zebrafish P450 17A1 mutant, and the

active site still resembled the structure in the other

proteins, with some important differences. These

P450 17A1 and 17A2 mutants had catalytic

profiles more similar to each other than did the

wild-type proteins. Docking with these structures

can explain several minor products, which require

multiple enzyme conformations. The 17a-

hydroperoxy (OOH) derivatives of the steroids

were used as oxygen surrogates. Human P450

1

7A1 plays a critical role in steroid metabolism,

converting both progesterone and pregnenolone to

their 17a-OH products (Fig. 1), which have

several roles. P450 17A1 also catalyzes 17a,20-

lyase (cleavage, or desmolase) reactions, with the

1

7A1 and zebrafish P450s 17A1 and P450 17A2

readily converted these to the lyase products in the

absence of other proteins or cofactors (with

1

ꢀ

P450 17A structure and functionꢀ

two 17a-OH steroids as substrates, yielding the

androgen androstenedione (Andro, from 17a-OH

the difference in lyase activity of P450s 17A1 and

1

7A2 to either of two possibilities: (i) either four

amino acid differences in the heme binding region

or a difference at residue 369 (Arg-369 in

zebrafish P450 17A1, corresponding to Arg-358 in

human P450 17A1 and Cys-358 in zebrafish P450

progesterone)

DHEA, from 17a-OH pregnenolone), which is

converted to Andro by a steroid dehydrogenase

3). At least 50 natural mutations in the enzyme

are known to cause clinical deficiencies (4, 5). On

the other hand, prostate cancer can be exacerbated

by androgens, and one therapeutic approach is to

target P450 17A1 to lower androgen production

and

dehydroepiandrosterone

(

1

7A2) are responsible for a change in the

mechanism, ablating lyase activity, or (ii) the X-

ray crystal structures are not revealing alternate

conformational states of P450 17A1 that are

needed for lyase activity (and are not available for

P450 17A2) (26). The zebrafish P450 17A1 Arg-

(

6, 7).

A number of questions in the field of P450

7A1 research have not been resolved. One

problem is that the catalytic specificity of P450

7A1 varies among species, in terms of the

3

69/human P450 17A1 Arg-358 site is particularly

1

interesting in that the human R358Q variant has

been known to lead to selective loss of the lyase

activity in patients for >20 years (18). This

phenomenon has been suggested to be due to a

1

balance between utilization of progesterone vs.

pregnenolone and between 17a-hydroxylation vs.

lyase activity (3, 8, 9). Cytochrome b₅(b₅)

stimulates P450 17A1 activity, and the extent to

which the lyase reaction is stimulated varies

among species, as well as the concentration of b₅

in steroidogenic tissues and their individual zones

selective loss of b binding, in that some lyase

5

activity could be restored by the addition of high

concentrations of purified b to human P450 17A1

5

expressed in a heterologous yeast system (19). The

results of both covalent crosslinking (27) and

NMR (28) studies also suggest an interaction of

(

3, 10, 11). Another controversy is the chemical

human P450 17A1 Arg-358 with b . However, the

5

mechanism of lyase reaction, for which both

3

+

same region has been implicated in binding of

NADPH-P450 reductase in NMR studies, and this

electrophilic

(Compound

I,

FeO )

and

nucleophilic (Compound 0, ferric peroxide,

2

+

protein and b compete (28, 29).

5

Fe O ¯) mechanisms have been proposed (12-17).

2

We now report zebrafish P450 17A1 and

A major question that we are trying to address is

the molecular basis for the selective loss of the

lyase activity, which is an inherent clinical

problem (3, 4, 18-20) but a goal in prostate cancer

therapy (21-24).

We have utilized a fish model discovered

by Zhou et al. (25). Teleost (bony) fish have two

genes, CYP17A1 and CYP17A2, which both code

P450 Subfamily 17A enzymes (~50% identical)

1

7A2 mutants in which we have made the

corresponding Arg/Cys substitution and the other

four residue changes based on the above

information (26) (supplemental Fig. S1). The X-

ray crystal structure of the five-residue P450 17A1

mutant was determined and was also utilized in

docking studies. We had previously shown that

human P450 17A1 could exhibit lyase activity in a

biomimetic model with the established “oxygen

surrogate” iodosylbenzene, which is considered to

generate either Compound I or a related species

(

supplemental Fig. S1). P450 17A2 catalyzes 17a-

hydroxylation of both progesterone and

pregnenolone but does not catalyze the lyase

reaction; zebrafish P450 17A1 catalyzes both 17a-

hydroxylation and the lyase activities, as in the

case of the mammalian enzymes (26).

Understanding the molecular basis of the lack of

lyase activity of zebrafish P450 17A2 may reveal

the basis of the lyase deficiency in humans. We

crystallized both zebrafish P450s 17A1 and 17A2,

in the presence of the inhibitor abiraterone (26),

but the structures of the active sites were very

similar to each other and to that of human P450

(

17). We have re-examined reactions with 17a-

OOH steroids, proposed early by Tan et al. (30-

2) to be intermediates in the lyase mechanism.

3

The 17a-OOH steroids were converted to lyase

products and hydroxylation products by human

P450 17A1 at very catalytically competent rates.

The 17a-OOH steroids were also converted to

lyase products by zebrafish P450s 17A1 and

1

7A2, as well as a human P450 R358C mutant that

is devoid of lyase activity in the usual assay

system. We interpret the 17a-OOH reactions in

1

7A1 bound to the same ligand (5). We attributed

2

ꢀ

P450 17A structure and functionꢀ

terms of a Compound I or related mechanism.

P450 17A2 and other normally lyase-inactive

P450 17A1 mutants appear to have inherent

features capable of lyase activity, but they are not

manifested in the normal catalytic reaction, even

product of 17a-OH progesterone (algestone,

supplemental Fig. S10). The corresponding site of

hydroxylation (16-) for 17a-OH pregnenolone was

identified by oxidizing the pregnenolone products

with cholesterol oxidase to form the 17a-OH

products mentioned above. In neither case could

we ascertain the stereochemistry at the 16-

position. Another product was identified as

ⁱⁿ^t^h^e^p^r^e^s^eⁿ^c^e^o^f^b5^.

RESULTS

6

b,16,17a-(OH)₃progesterone by comparison

Identification of more reaction products of

zebrafish P450 17A1 and 17A2

(

HPLC, MS) with the product formed by human

P450 17A1 (17).

In our previous work with zebrafish P450

1

7A2 (33), we reported only the 17a-OH steroids

Site-directed mutagenesis

formed from progesterone and pregnenolone and

concluded that they were final products. Zebrafish

P450 17A1 formed the lyase products Andro and

DHEA from 17a-OH progesterone and 17a-OH

pregnenolone, respectively. P450 17A2 did not

form any lyase products with either 17a-OH

steroid (33).

We increased the enzyme concentrations

and reaction time and identified more products,

including 17a,21-(OH)₂progesterone (11-

Although zebrafish P450 17A1 and 17A2

are only ~ 50% identical (Fig. 2), their 3-

dimensional structures are very similar,

particularly in the substrate binding region (33).

Thus, superimposition of the zebrafish P450 17A1

and 17A2 complexes with abiraterone results in a

root mean square deviation (r.m.s.d.) between the

two models of 1.49 Å (see Fig. 14C, 14D in (33)),

with only minimal differences in the active sites.

We considered the residue Arg-358

deoxycortisol),

16,17a-(OH)₂

6b,16,17a-(OH)₃

progesterone

progesterone,

(numbering system of human P450 17A1). This

amino acid is Arg in both zebrafish (Arg-369) and

human P450 17A1 (Arg-358) but is Cys (358) in

zebrafish P450 17A2 (33) (supplemental Fig. S1).

Because the human P450 17A1 mutation R358Q

results in preferential loss of lyase activity

compared to 17a-hydroxylation (3, 18-20) we

tested the hypothesis that changing this single

residue to arginine could generate lyase activity in

zebrafish P450 17A2.

(

algestone),

1

7a,21-(OH)₂pregnenolone, and 16,17a-(OH)₂

pregnenolone from both zebrafish P450 17A1 and

7A2 (Fig. 2). These were identified by their mass

1

spectra and by co-chromatography with the

products we characterized in oxidations catalyzed

by human P450 17A1 (17) plus a commercial

sample

of

11-deoxycortisol

(17,21-(OH)₂

progesterone) (supplemental Fig. S9).

^H^u^m^aⁿ^P⁴⁵⁰¹⁷^A¹^R³⁵⁸^C^h^a^d^sⁱ^mⁱ^l^a^r^kcat

Two products derived from 17a-OH

progesterone correspond to hydroxylation at the 21

and 16 positions. The 21-hydroxy product had not

been identified in our previous work with human

P450 17A1, but 17a,21-(OH) progesterone (11-

deoxycortisol) co-chromatographed with

commercial standard and had a very similar mass

spectrum (supplemental Fig. S9). In particular, the

m/z 317 peak corresponds to the loss of –CH OH

and K values and catalytic efficiency (k /K ) for

m

cat

m

progesterone 17a-hydroxylation compared to the

wild-type enzyme (Table 1). Human P450 17A1-

R358C had no detectable lyase activity with either

2

1

7a-OH progesterone or 17a-OH pregnenolone in

a

the absence or presence of b . The zebrafish P450

5

17A1-R369C mutant had only 3% of the 17a-OH

progesterone lyase activity of the wild-type

enzyme (supplemental Fig. S2) but retained ~1/3

of its progesterone 17a-hydroxylation catalytic

efficiency (Table 1). Zebrafish P450 17A2-C358R

also had reduced 17a-hydroxylation efficiency

(~1/3) (Table 1) but no detectable lyase activity

2

and the loss of the elements of H O (probably

2

1

7a-OH) from this yields m/z 299 (positive ion

APCI MS) (34). P450 17A1-catalyzed 21-

hydroxylation activity of a steroid possessing a 3-

4

,5

keto-D skeleton has been observed previously in

the 21-hydroxylation of progesterone by human

P450 17A1 (Fig. 2) (35). A similar approach was

used in the identification of the 16-hydroxylation

with

17a-OH

progesterone

or 17a-OH

pregnenolone.

3

ꢀ

P450 17A structure and functionꢀ

Previous X-ray crystallography studies

showed no major differences between residues in

the substrate binding sites of wild-type zebrafish

P450s 17A1 and 17A2 (33). However, four

differences were observed in the heme binding

region (supplementary Fig. S1). In addition to

switching the Arg/Cys pair at positions (Arg) 369

progesterone

respectively). These differences in the b effect

indicate the complex nature of how b affects the

lyase activity of P450 17A1 (i.e., b interaction

with P450 17A1 is not just related to a single

amino acid residue).

Wild-type zebrafish P450 17A2 and its

five-residue mutant (DVRLL) were also analyzed

for the three hydroxylated products of 17a-OH

progesterone and 17a-OH pregnenolone (Fig. 3C,

and

17a-OH

pregnenolone,

5

(

17A1) and (Cys) 358 (17A2) (Table 1), we added

these four substitutions (zebrafish P450 17A1:

D309E, V321T, L372M, L407M; 17A2: E298D,

T310D, T310V, M361L, M396L), with the

hypothesis that making the changes could

collectively convert the activity of P450 17A1 to

that of P450 17A2 and vice-versa (Fig. 3;

supplemental Table S1, Figs. S3-S5). As

previously reported (33), wild-type P450 17A2, in

3

D; supplemental Table S2, Figs. S6-S8), i.e., the

1

6- and 21-hydroxy and 6b,16-dihydroxy products

(

see above). The five mutations all increased the

catalytic efficiencies for all three hydroxylations

Fig. 3C, 3D, supplemental Table S2, Figs. S6-

(

S8). The catalytic efficiencies of the

hydroxylations were not so strongly affected by

the presence of zebrafish b , has higher catalytic

5

efficiency for 17a-hydroxylation than P450 17A1

with both the substrates progesterone and

pregnenolone (Fig. 3A, 3B; supplementary Table

S1, Fig. S3). Making the five substitutions

the addition of b (Fig. 3C, 3D).

5

X-ray structure of the zebrafish P450 17A1-

ETCMM mutant

(

ETCMM) increased the 17a-hydroxylation

catalytic efficiency of zebrafish P450 17A1 4- to

-fold with both progesterone and pregnenolone as

To allow comparisons with the wild-type

zebrafish P450 17A1 enzyme (33), we determined

the crystal structure of the zebrafish P450 17A1-

ETCMM mutant protein in the presence of the

drug abiraterone at 3.03 Å resolution (Fig. 4,

Table 2). As in the case of the wild-type protein

complex with inhibitor, crystals of the ETCMM-

mutant complex with abiraterone were grown

using in situ proteolysis with subtilisin (37).

However, unlike the crystallization experiments

with wild-type zebrafish P450 17A1 that were

conducted at room temperature, crystals for the

6

substrates (Fig. 3B). This was the case with or

without b ; the presence of b reduced the 17a-

5

hydroxylation catalytic efficiencies several fold

supplementary Table S1). Conversely, making the

P450 17A1-like mutations in zebrafish P450 17A2

DVRLL) decreased the 17a-hydroxylation

(

catalytic efficiencies several-fold toward both

progesterone and pregnenolone (Fig. 3A, 3B,

supplemental Table S1). The catalytic efficiencies

for the lyase activity were measured for the P450

mutant were grown at 4 °C and their space (P4 22)

1

7A1 mutant ETCMM (Fig. 3B; supplemental

differs from that of the wild-type protein (P3 21).

1

Table S1, Fig. S4) with and without b : 0.06 (-b )

The structure of the mutant protein complex was

solved by molecular replacement using the

coordinates of one of the two wild-type zebrafish

P450 17A1 protein molecules minus water and

ions as search model. As in the case of the wild-

type protein crystals, the tetragonal zebrafish P450

5

-

1

-1

and 1.1 (+b ) µM min and 0.17 (-b ) and 0.47

5

-

1

-1

(

+b ) µM min for 17a-OH progesterone and

5

1

7a-OH pregnenolone, respectively. The effects

of b stimulation of lyase activity were ~18-fold

5

and ~2.8-fold, respectively, for 17a-OH

progesterone and 17a-OH pregnenolone, which

contrasts with the case of wild-type zebrafish P450

1

7A1-ETCMM mutant crystals contained two

complexes per asymmetric unit. Molecule B could

be completely traced with no interruptions in the

chain between residues 39 and 508, and in

molecule A only four amino acids (176-179) in the

loop region between helices D and E are missing

in the final model (Fig. 4A). Thus, a total of 935

residues from two complexes are included in the

final model of the zebrafish P450 17A1-ETCMM

1

7A1 (Fig. 3B; supplemental Table S1). In the

wild-type P450 17A1, the b stimulation of lyase

5

activities was consistent between 17a-OH

progesterone and 17a-OH pregnenolone, with 3.5-

fold stimulation for both substrates (0.43 to 1.5

-

1

-1

and 0.78 to 2.7 µM min for 17a-OH

4

ꢀ

P450 17A structure and functionꢀ

structure (Table 2) as compared to 828 residues in

the crystal structure of the wild-type protein (33).

Whereas the C-terminal region is resolved up to

residue 508 in both the wild-type and mutant

crystals, the N-terminal region features an

additional 44 residues in the latter (Fig. 4A). A

shared property of the two crystals is the formation

of a dimer that is stabilized by two disulfide

bridges involving Cys-146 and Cys-437. The two

independent molecules A and B in the zebrafish

P450 17A1-ETCMM mutant crystal structure

exhibit rather similar conformations with a root

mean square deviation (r.m.s.d.) of only 0.67 Å for

of the vinyl moiety of heme (3.6 Å distance

between the Og1 of threonine and the heme

methylene carbon) is well tolerated. Val-377,

adjacent to Thr-321, is slightly displaced relative

to its position in the wild-type structure next to

Val-321 (Fig. 5C). The distance between the Og1

of Thr-321 and Cg2 of Val-377 in the mutant

structure (4.2 Å) is slightly longer than the

corresponding distance between the Cg2 of Val-

3

21 and Cg2 of Val-377 in the structure of the

wild-type protein (3.7 Å). The closest distance

between Thr-321 (Cg2) and Met-372 (Cg; mutated

from leucine) is 4.3 Å (Fig. 5C). The distance

from the methyl carbon of the threonine to the

methionine sulfur is 5.1 Å. The closest distance

between the side chains of Val-321 and Leu-372 in

the wild-type structure is rather similar, 4.2 Å,

again indicative of only minor adjustments of

residues that line the active site as a consequence

of the mutations.

4

65 Ca pairs (Fig. 4C). Conversely, the r.m.s.d.

between the wild-type and mutant P450 17A1-

abiraterone complexes is significantly higher

(

molecules B) and amounts to 2.16 Å for 415 Ca

pairs. Therefore, the structures of the zebrafish

P450 17A1 and 17A2 complexes with abiraterone

exhibit a closer similarity (r.m.s.d. of 1.49 Å; vide

supra) (33) than those of the zebrafish P450 17A1

wild-type and ETCMM mutant complexes.

Compared to the above D309E, V321T,

and L372M mutations, the R369C mutation is

more drastic in that it replaces a longer, polar side

chain with a short hydrophobic one and removes a

positive charge. Remarkably, this mutation alone

nearly abolished the lyase activity of zebrafish

P450 17A1 (supplemental Fig. S2). In the

structure of the abiraterone complex, the R369C

mutation results in significant conformational

adjustments of two neighboring side chains and

the insertion of a water molecule that occupies a

portion of the space vacated by the arginine side

chain (Fig. 5D; in complex A the position of this

water is occupied by a chloride ion). Mutation of

Arg-369 flips the side chains of Glu-365 and Arg-

Four of the five mutated residues are ~ 10

Å away from the iron center in the heme prosthetic

group (Glu-309, Val-321, Met-372, Met-407), and

Cys-369 is farther removed (ca. 15 Å) (Fig. 5A).

The overlay of the wild-type and mutant complex

structures demonstrates that the active site regions

exhibit closely similar main and side chain

conformations in the majority of cases. The side

chain of Glu-309 is more extended as a

consequence of the additional methylene group

relative to aspartate in the P450 17A1 wild-type

protein. As a result, the side chain of the adjacent

Asp-124 shifts and rotates slightly, and the

shortest distance between carboxylate oxygens of

the two is 2.6 Å in the mutant complex (dashed

line, Fig. 5B). The closest distance between the

side chains of Asp-309 and Asp-124 in the wild-

type complex is identical and thus the mutation

does not appear to create a clash. No basic residue

is situated within a close range from these two

residues, and it is possible that one of the

carboxylates is protonated to prevent a Coulombic

repulsion.

3

58 around (the latter of which corresponds to the

conserved Arg-347 of human P450 17A1, known

to also be critical to lyase function (20)), such that

the latter two engage in a salt bridge in the mutant

structure. In addition, the guanidino moiety of

Arg-358 also forms a H-bond to the water newly

present at this site. In the structure of the wild-type

protein complex, the head group of Glu-365 is

inserted between the guanidino moieties of Arg-

3

69 and Arg-358 (dashed lines, Fig. 5D), but the

The Val-321 in wild-type P450 17A1 and

Thr-321 in the mutant protein superimpose neatly

Fig. 5C), indicating that the two residues are

interchangeable and insertion of a hydroxyl in

place of the native methyl group in close vicinity

distances are too long (> 4 Å) to allow formation

of salt bridges. Instead, the particular arrangement

of the three amino acids produces electrostatically

favorable interactions that tie together helices J´

and K (Fig. 5D). Thus, the R369C mutation may

(

5

ꢀ

P450 17A structure and functionꢀ

affect the relative mobility of these helices, one of

which cradles heme (K). The side chain of Lys-

reaction cannot be universal in its occurrence

among P450 17A1 enzymes. Thus, the previous

studies with 17-O-acetyl testosterone (38, 39)

4

60 from a third helix (L) that juts into the region

affected by the R369C mutation also alters its

conformation slightly in the mutant structure

relative to the situation in the wild-type complex.

However, a salt bridge between Lys-460 and Glu-

cannot be used to support a general FeO ¯ Baeyer-

Villiger mechanism (Fig. 6).

2

Reaction of 17

P450 17A enzymes

7a-OOH pregnenolone and 17a-OOH

a

-hydroperoxy steroids with

4

56 is conserved in both structures (Fig. 5D).

Residues of the K and L helices also interact with

helix I, which spans one side of the active site

1

progesterone have both been synthesized

previously (41-43) and considered as possible

intermediates in the lyase reactions (30-32),

although these proposals can no longer be

considered viable in light of current information

about the catalytic mechanism (16, 17, 44).

However, Tan and associates had reported the

rearrangement of the 17a-OOH steroids to lyase

products using crude tissue extracts (30-32). We

synthesized both 17a-OOH pregnenolone and

(

Fig. 4A, 4B). Panel D in Fig. 5 also illustrates,

together with panel C, how the L372M mutation

slightly pushes away the Phe-446 phenyl ring (the

methionine sulfur stacks onto the edge of the

aromatic moiety).

The L407M mutation is more benign

compared to the above replacement of arginine by

cysteine. Consistent with the similar spatial

requirements and hydrophobic nature of leucine

and methionine, the mostly hydrophobic residues

1

7a-OOH progesterone (supplemental Figs. S11,

S12) and added these biomimetic reagents to P450

7A enzymes, without NADPH or reductase, in

(

4

Leu, Ile, Val, Pro, and His) surrounding residue

07 show only minor adjustments in their side

1

chain conformations (Fig. 5E). The closest

distance between Met-407 and His-411 is 4 Å

order to test the rearrangement hypothesis

proposed by Tan et al. (32) (Fig. 7).

(

S…Ne2). Importantly, in terms of the vicinity of

The same products were observed with

the amino acid at position 407 and heme, the

closest distances between Leu-407 and heme in

the wild-type structure and Met-407 and heme in

the mutant structure are very similar (4.7 and 4.5

Å, respectively).

human P450 17A1 (Fig. 8A, 8B) and with

zebrafish P450s 17A1 and 17A2 (supplemental

Fig. S15). Major products included the 17a-OH

products, the lyase products (Andro and DHEA),

and

supplemental Fig. S15). The identities of DHEA

and Andro (Fig. 8B) were confirmed by LC-MS

the

16,17-(OH)₂

steroid

products

(

Lack of conversion of progesterone to 17-O-

acetyl testosterone

One piece of evidence offered for a ferric

peroxide (Compound 0) mechanism is the reported

conversion of progesterone to 17-O-acetyl

testosterone by porcine testicular P450 17A1 (Fig.

(

supplemental Figs. S13, S14).

The lyase reactions were rapid with

human P450 17A1 (without NADPH or

-1

reductase), with initial rates of > 120 min (Fig.

8

C, 8D), more than fast enough to be catalytically

6

) (38, 39). Such a Baeyer-Villiger reaction is a

-

1

competent (cf. k_c_a_t4.3 and 1.1 min for 17a-OH

progesterone and 17a-OH pregnenolone in the

normal NADPH-supported reactions, respectively)

derivative of the proposed FeO ¯ mechanism (12)

2

and has also been proposed for the P450 51A1

lanosterol 14a-demethylation reaction (40). We

conducted UPLC-UV assays with human P450

(

36). No further acceleration of the reactions was

seen with b (Fig. 8C), which is very stimulatory

5

1

7A1 and progesterone (under the usual conditions

in the normal NADPH-P450 reductase-supported

reaction (36, 45) but apparently without the

involvement of electron transfer (12, 46).

with NADPH-P450 reductase, b , NADPH, and

5

O ) and could not detect any 17-O-acetyl

2

testosterone (separable by HPLC), at a level of

With both 17a-OOH pregnenolone and

<

0.15% of the 17a-OH progesterone formed (rate

-

1

17a-OOH progesterone, the lyase reactions were

observed with zebrafish P450 17A2 (supplemental

Figs. S15, S16). These had never been shown in

the NADPH-P450 reductase-supported reaction

of formation <0.0013 min , corresponding to less

than one turnover/13 hours). Porcine P450 17A1

was not available for direct comparison, but this

6

ꢀ

P450 17A structure and functionꢀ

(

33), even in the presence of b . In other

to 17a-hydroxylation, are able to form the 16- and

5

experiments (data not presented), the human P450

7A1-R358C and zebrafish P450 17A1-R369C

2

1-hydroxylated

products

from

17a-OH

1

progesterone and 17a-OH pregnenolone, plus

mutants, devoid of lyase activities in the usual

NADPH-supported reactions, showed lyase

activity with both 17a-OOH pregnenolone and

6

b,16,17a-(OH) progesterone.

3

The five mutations we made based on the

crystal structures (26) rendered zebrafish P450

7A1 and 17A2 more similar to each other. The

1

7a-OOH progesterone. This was not stimulated

1

by the addition of b . The rates of formation were

5

mutations increased the 17a-hydroxylation

activity of zebrafish P450 17A1 and reduced the

1

The changes also decreased the lyase activity of

P450 17A1 (Fig. 3A, 3B; supplemental Fig. S4,

Table S1). The R369C mutation nearly ablated the

lyase activity of zebrafish P450 17A1

~

½ of those observed for the wild-type enzymes.

We also saw similar lyase activity with the

zebrafish P450 17A2-C358R mutant.

7a-hydroxylation activity of P450 17A2 (Fig. 3).

None

of

the

observed

products

corresponded to 21-hydroxylation, i.e. 17a-, 21-

(

^O^H⁾2

progesterone

(11-deoxycortisol)

or

pregnenolone

(11-deoxycorticosterone),

the

(

supplemental Fig. S2) but the inverse change

C358R) in P450 17A2 (supplemental Fig. S1) did

former of which had been reported in the reaction

of 17a-OOH progesterone with bovine adrenal

cortex microsomes (31) (and was a product of

not result in any detectable lyase activity. Further,

the five amino acid changes (P450 17A2-DVRLL)

did not impart lyase activity.

1

7a-OH steroids in the usual NADPH-dependent

assay (Fig. 3C, supplemental Table S2)). The

major unidentified products (supplemental Fig.

S15) all had mass spectra with the same parent

ions as the 17a-OOH steroids that they were

derived from and are proposed to be hydroxylated

The structure of zebrafish P450 17A1-

ETCMM determined with crystals grown at 4 °C

and in the presence of subtilisin yielded a model

with more than 40 additional amino acids on the

N-terminal side compared to the structure of wild-

type zebrafish P450 17A1 (26). The region

between residues 39 and 82 entails a 22-residue

loop, an a-helix with three full turns, and a six-

residue b-strand. The latter forms an antiparallel

sheet with amino acids 84 to 90, which were

present in the previous model (Fig. 4A). The likely

reason for the different length of the protein

trapped in crystals of zebrafish P450 17A1-

ETCMM is that in situ proteolysis with subtilisin

progressed more slowly at the lower temperature

(wild-type crystals had been grown at room

temperature). Loop regions that were absent in the

model of the wild-type zebrafish P450 17A1

protein could be completely built in the case of

complex B in the structure of the mutant. Missing

loops in the former were not a consequence of

subtilisin action but probably could not be traced

in the electron density owing to locally increased

mobility of the chain (the resolution of the wild-

type zebrafish P450 17A1 structure was also

somewhat lower (26)). In addition, altered packing

interactions resulting from the difference in space

group symmetry between the zebrafish wild-type

and mutant P4540 17A1 may have resulted in

some loop regions becoming more ordered.

However, the formation of two disulfide bridges

1

7a-OH steroids. Attempts to define the structures

of the three major peaks have not been successful

yet due to the limited amount of material; we

obtained NMR spectra of three peaks, and in two

of these the chemical shift of the 18-CH protons

3

was shifted downfield but we were unable to make

an unambiguous assignment.

Conceivably

epoxides could also be derived from the

unsaturated moieties in the steroids or,

alternatively, the structures of these products could

be rationalized as hemiketals (which would form if

the 17-hydroperoxide bond cleaved and either the

C17-C16 bond or the C17-C13 bond were cleaved

to generate a C17-radical that can oxygen rebound

to the Compound II iron hydroxide intermediate).

DISCUSSION

One of the most interesting questions

about P450 17A1 is the basis of the 17a,20-lyase

activity, and a major issue is why some single base

variants selectively lose this activity (3, 18, 20).

We have been studying zebrafish P450 17A2 as a

means of addressing the problem. We now show

that zebrafish P450s 17A1 and 17A2 have

multiple hydroxylation activities and, in addition

7

ꢀ

P450 17A structure and functionꢀ

between the two independent abiraterone

complexes per crystallographic asymmetric unit is

a feature that is conserved in the zebrafish wild-

type P450 17A1 and P450 17A1-ETCMM mutant

structures. Overlays of the structures of the

abiraterone complexes of zebrafish wild-type

P450s 17A1 and 17A2 (26)—and wild-type P450

obviously have a significant effect on their

hydroxylation activities.

By comparison, residue 369 (zebrafish

P450 17A1-R369C mutant) has no direct

interaction with heme and yet it causes loss of

lyase activity with zebrafish P450 17A1

(supplemental Fig. S2) (also true with human P450

17A1 Arg-358). The arginine sits adjacent to the

junction of helices J´, K, and L (Fig 5D), whereby

the two latter stabilize heme and interact with the

long I helix that forms one wall of the active site.

Lys-460, from helix L, is directly adjacent to Arg-

369 and is just six residues downstream from Cys-

453, which is coordinated to the iron center.

Although the arginine is farther removed from

heme than the other four mutated residues, it

appears to nevertheless occupy a key position, and

its mutation to cysteine changes the electrostatics

at a three-helix junction. Thus, the mutation may

affect the relative mobility of these helices and

most likely also alter the dynamics of the

interaction of helices K and L with helix I, which

forms a backdrop for the substrates. Importantly,

the corresponding arginine in human 17A1 (Arg-

358) was mapped to the binding region with b₅

(47). Indeed, inspection of a computational model

of the human 17A1:b₅complex based on a

crosslinking/mass-spectrometric analysis (27, 48),

reveals that Arg-358 is in proximity to Ser-43,

1

7A1 and P450 17A1-ETCMM (this work)—

reveal a smaller deviation between the former pair.

Despite these conformational differences

in the overall structures of zebrafish wild-type

P450 17A1 and the ETCMM quintuple mutant

(

r.m.s.d. of 2.16 Å), the active site regions exhibit

only minor differences (Fig. 5A). Two of the

mutations are rather conservative (L372M and

L407M), and inspection of the structural

adjustments in the immediate vicinities of the

newly introduced methionines confirms that these

are indeed rather minor (Fig. 5C, 5D, 5E). Even in

the cases of the D309E (Fig. 5B) and V321T (Fig.

5

1

C) mutations, comparison of the zebrafish P450

7A1 and ETCMM mutant structures reveals no

significant movements of these residues or

formation of novel interactions. The longer

glutamate side chain at position 309 is

accommodated by a slight repositioning of the

carboxylate moiety from Asp-124, without loss of

the close approach between the two. Similarly, the

orientation of the Thr-321 side chain barely

changes relative to that of Val-321 in the wild-type

structure. Nevertheless, introduction of these four

residues that are all some 10 Å or less away from

Glu-68, and Glu-69 of b (supplemental Fig. S17).

5

Therefore, beyond stabilizing internal interactions

in 17A1, Arg-369 (h17A1:Arg-358) sits at the

3

+

the Fe center (shortest contact involving a side

interface with b that enhances the lyase activity.

5

chain atom; Fig. 5A) from the zebrafish P450

The R®C mutation would be expected to

1

7A2 into the P450 17A1 protein improves the

significantly alter the 17A1:b binding interface.

5

hydroxylation activity of the latter. In turn,

transplanting the four 17A1 residues at positions

In our earlier study on zebrafish P450 17A

enzymes (26), we did not find additional products,

other than those depicted in Fig. 1. Secondary

products were seen here, similar to those observed

with human P450 17A1 (17, 36), after raising the

enzyme concentrations and reaction time (Fig. 2).

These are minor products, as seen by the lower

rates of formation (Fig. 3C, 3D; supplemental

Table S2), and we do not know if any have

physiological activities. 16a-OH progesterone has

been detected in vivo in human serum (49). We

used molecular mechanics to calculate models of

the complexes between zebrafish P450 17A1-

ETCMM and mono-, di-, and trihydroxy

progesterones as well as the mono- and dihydroxy

3

09, 321, 372, and 407 into the P450 17A2 protein

lowers the hydroxylation activity of the latter.

Clearly, the proximity of these amino acids to the

heme group affects the relative activity that is

shared between the P450 17A1 and 17A2

enzymes. The static image provided by the

structures may not allow us to pinpoint the actual

reason(s) for the observed changes in

hydroxylation activity. More subtle differences in

direct contacts to heme atoms, caused by the four

above mutations that may affect the iron center

and possibly have consequences also at the level

of the dynamic behavior of the two enzymes,

8

ꢀ

P450 17A structure and functionꢀ

pregnenolones (Fig. 9). In all cases, these

substrates can easily be accommodated in the

active site, using as guides the orientations of

abiraterone in zebrafish P45017A1 (wild-type and

ETCMM mutant; this work, Fig. 9A) and

progesterone in the wild-type zebrafish P450 17A2

complex structures (26) as well as the structures of

human P450 17A1-substrate complexes (50).

Taking into consideration sterics and H-bond

interactions, all manually built models could be

quickly relaxed with the Amber force field in the

UCSF Chimera suite (51) (see Experimental

Procedures) and relying on repeated rounds of

steepest descent and conjugate gradient energy

minimization approaches. In particular, the 16a-

OH oxygen in the dihydroxy products assumes a

position that lies only moderately farther way from

explain the lyase reactions. However, like

mechanism A, it does not explain the 17a-OH

products or the hydroxylated 17a-OH steroids

(

e.g., 16-OH and others, supplemental Fig. S15).

Compound II would not be expected to do

methylene hydroxylations. Mechanism B involves

heterolytic cleavage of the hydroperoxide to form

Compound I. Mechanism Ba readily explains 16-

i

hydroxylation. Mechanism Ba can explain the

ii

lyase reaction. A test for this hypothesis would

involve using 16-d₂17a-OOH steroids and

checking for loss of deuterium label in the

DHEA/Andro formed, but we have not prepared

this material (a similar experiment could be done

with 16-d₂steroids in the normal NADPH-

supported reaction). Mechanism Bb involves

abstraction of a hydrogen atom from the alcohol of

the 17a-OH steroid. Bonomo et al. (52) have

argued against such a mechanism based on density

functional theory calculations (with human P450

17A1). Mechanism Bc involves nucleophilic

attack of the 17a-OH oxygen on Compound I (not

considered in Bonomo et al. (52)), as proposed in

Fig. 7, plus a dioxetane intermediate originally

proposed by Tan and Rousseau (32) (mechanism

A, also Fig. 7). In principle, the dioxetane

mechanism should yield chemiluminescence (53,

54). We have not looked for chemiluminesence;

the expected energy yield would be expected to be

both low and transient.

3

+

Fe than the 17a-OH oxygen: 4.5 vs. 4.0 Å

progesterone) and 4.9 vs. 4.4 Å (pregnenolone),

(

respectively. As well, in the case of the trihydroxy

progesterone product, the 6b-OH oxygen is

situated 5 Å from the metal ion. Therefore, these

models are qualitatively consistent with the

experimentally established formation of additional

products catalyzed by the zebrafish P450 17A1

wild-type and ETCMM mutant enzymes.

1

7a-OOH steroids have previously been

considered as intermediates in androgen synthesis

30-32) but there is no evidence that these are

(

formed, and a dioxygenase reaction to generate

them has never been reported. Nevertheless, the

original conclusions reached about P450-catalyzed

rearrangements of the synthetic hydroperoxides

We propose that the products are

3

+

dependent upon Compound I (FeO ) chemistry,

and our overall results imply that Compound I—or

an equivalent—is formed and has the capability of

producing the lyase reactions. Thus, these results,

coupled with our earlier work with the oxygen

surrogate iodosylbenzene (17), provide evidence

that P450 17A1 Compound I (which must be

made, to rationalize the many hydroxylation

reactions (Fig. 11)) is capable of lyase reaction

chemistry. However, our work, as mentioned

earlier (17), does not indicate the extent to which

(

done with crude beef adrenal and rat testis

preparations) appear to be valid (30-32). The rates

of formation of the lyase products (Fig. 8C, 8D,

supplemental Fig. S16) are more than fast enough

to be consistent with mechanistic interpretations,

which can be raised as a criticism of our earlier

biomimetic work with iodosylbenzene (17).

1

7a-OH steroids and several hydroxylated

1

7a-OH steroids were generated in the 17a-OOH

3

+

2+

–

Compound I (FeO ) and Compound 0 (Fe O )

reactions (supplemental Fig. S15). These are

relevant to the mechanism of the 17a-OOH

reactions and are not explained by mechanism A

or C in Fig. 15. In mechanism A (Fig. 10), the

only products would result from lyase cleavage

and neither 17a-OH nor other hydroxylated

steroids are explained. Mechanism C, involving

homolytic cleavage of the hydroperoxide, can

2

reactions contribute to the normal enzymatic lyase

reaction.

We conclude that P450 17A2 has a

conformation such that it cannot position the 17a-

OH group for proper attack by Compound I or

Compound 0; i.e., a conformation to facilitate the

reactions is not available to P450 17A2. However,

9

ꢀ

P450 17A structure and functionꢀ

a possible conformation(s) must exist for the

productive interaction of the heme ferric iron with

the 17a-OOH steroids to generate Compound I

radicals (56). However, the generation of

hydroxylated steroid products argues against the

involvement of alkoxy radicals, which would be

unlikely to do such chemistry. We conclude (Fig.

(

Fig. 9). b is not needed to facilitate this latter

5

1

1), in support of our earlier studies with oxygen

biomimetic reaction, but in the normal 17a-OH

surrogate iodosylbenzene (17), that P450 17A

enzymes can use Compound I chemistry (Fig.

steroid/NADPH-based reaction even the presence

of b is not sufficient to yield lyase activity for

5

1

1B, 11C) to perform lyase reactions. However,

P45017A2 or the P450 17A2-DVRLL mutant.

Overall, we conclude that zebrafish P450s

our results cannot preclude involvement of

Compound 0 (Fig. 16A) in the normal NADPH-

based reaction.

Why do zebrafish P450 17A2 and human

P450 Arg-358 mutants (Table 1, (18-20)) not

catalyze lyase reactions? At least with P450 17A2,

the reason is not only the lack of one residue just

not being arginine (Cys-358) (supplemental Fig.

S1). Substitution at that position has a more

general effect on the structure, as discussed earlier

1

7A1 and 17A2, like human P450 17A1 (17),

form a number of minor hydroxylation products

Fig. 2). Productive juxtapositions in the active

site can be rationalized in the crystal structure

Fig. 9). Site-directed mutations of zebrafish

(

P450s 17A1 and 17A2, based on their crystal

structures (26), alter the catalytic selectivity of the

enzymes to be more like each other (Fig. 3;

supplemental Fig. S1, Table S1). However, none

of the mutations (supplemental Fig. S1) yielded

lyase activity with P450 17A2 (supplemental

Table S1). The effects of the four substitutions

near the heme periphery were not as dramatic as

those resulting from mutation of a more distant

arginine (Arg-358 in human P450 17A2, Arg-369

in zebrafish P450 17A1). However, changing Cys-

(

Figs. 4, 5), and substituting one residue is not able

to overcome a basic defect in the enzyme.

However, the lyase-defunct enzymes are able to

utilize the 17a-OOH steroids in a manner

somewhat distinct from the normal reaction (Fig.

7). We have already concluded (36) that multiple

conformations of P450 17A1 are involved in

catalysis, in agreement with Scott and associates

(22, 47), who have defined structural variants

found both in the absence and presence of ligands

in NMR and X-ray crystallography studies.

Zebrafish P450 17A2 is capable of lyase activity

with the 17a-OOH steroids in that it can achieve

an appropriate conformation that appears to be

precluded in the conventional reaction.

3

58 in zebrafish P450 17A2 to arginine did not

impart lyase activity. The interpretation of these

site-directed mutagenesis studies is complex in

that the Arg ® Cys mutation attenuated

progesterone 17a-hydroxylation activity in

zebrafish P450 17A1 by ~ 2/3 but so did the

opposite mutation in zebrafish P450 17A2; the

analogous mutation had no effect on progesterone

1

7a-hydroxylation activity catalytic efficiency in

human P450 17A1 (Table 1).

Work with the biomimetic model 17a-

EXPERIMENTAL PROCEDURES

Chemicals

OOH steroids showed facile conversion to

multiple products, especially the lyase products,

with both 17a-OOH progesterone and 17a-OOH

pregnenolone (Fig. 8; supplemental Figs. S15,

S16). The lyase products were formed even with

P450s that did not have this capability in the

normal NADPH-supported reactions. What does

this mean? We interpret the results in the context

Synthesis of 17 -OOH pregnenolone

a

Preparation of 17a-OOH pregnenolone

was done as previously described (43).

Pregnenolone (316 mg, 1.0 mmol) was dissolved

in anhydrous benzene (50 ml) and tetrahydrofuran

(

6 ml) in a Parr pressure reaction vessel.

3

+

Potassium superoxide (284 mg, 4.0 mmol) and 18-

crown-6 (449 mg, 1.7 mmol) were added and the

vessel was closed securely. The vessel was

pressurized with 8 atmospheres of O gas (~130

psi, from a cylinder) and the mixture was stirred at

of formation of Compound I (FeO ), which can

proceed to yield all of the observed products (Fig.

1

0B, 10C). There are important caveats in the use

of alkyl hydroperoxides as oxygen surrogates (55),

in that some of the reactions observed with these

can be attributed to reactions involving alkoxy

2

6

°C for 4 hours. The reaction was quenched with

1

0

ꢀ

P450 17A structure and functionꢀ

cold 10% HCl (10 ml, aq). The product was

extracted with diethyl ether, the organic phase was

was added and the mixture was continued to stir

for a total of 4 hours. The reaction was diluted

washed once with H O and dried over anhydrous

with H O (500 ml) and extracted with ethyl

2

MgSO , and the solvent was removed using a

acetate. The organic phase was washed twice with

4

rotary evaporator. The crude product (17a-OOH

pregnenolone) was purified by semi-preparative

HPLC: a 250 mm ´ 10 mm Phenomenex

octadecylsilane C18 (5 µm) column was used at a

H

2

O, followed by brine, and dried over anhydrous

MgSO . The solvent was removed using a rotary

evaporator. The crude solid was dissolved in warm

CH OH (12 ml) and glacial acetic acid was added

4

3

-

1

(

12 ml) to cleave the 3-O-ethoxy group. The

flow rate of 3 ml min with a linear gradient of

0-70% B (v/v) over 40 minutes (solvent A was

H O and solvent B was CH CN). A portion of the

mixture was stirred at room temperature for 3

hours, concentrated using a rotary evaporator, and

poured into H O to form an off-white precipitate.

The crude product (17a-OOH progesterone) was

purified by silica gel chromatography (20% ethyl

acetate in hexanes, v/v). The product was further

purified with a semi-preparative 250 mm ´ 10 mm

Phenomenex octadecylsilane C18 column (5 µm,

linear gradient of 40 to 70% (v/v) over 40 min;

solvent A was H O and solvent B was CH CN).

4

2

3

product (t_R21 min) was collected (in

2

1

approximately 4% overall yield). H NMR (400

MHz, d -DMSO, supplemental Fig. S11) δ 5.27

6

(

d, 1H), 4.59 (d, 1H) 3.29-3.22 (m, 1H), 2.45-2.38

1H), 2.17 (s, 3H), 2.14-2.08 (overlapped, 2H),

1

(

0

.99-1.88

overlapped, 13H), 1.02-0.84 (overlapped, 2H),

.92 (s, 3H), 0.54 (s, 3H) (57) (see also (58-60)).

(overlapped,

2H),

1.77-1.16

2

3

+

ESI HRMS: calculated for C H O [MH -18] m/z

The product (t_R22 min) was collected in

approximately 12% overall yield. H NMR (400

2

1

33

4

1

3

31.2268; found 331.2275 (D 2.1 ppm).

Treatment of the product

with

MHz, d

1H), 2.45-2.35 (m, 3H) 2.25-2.13 (m, 2H), 2.17 (s,

H), 1.99-1.88 (overlapped, 2H), 1.81-1.55

overlapped, 9H), 1.44-1.34 (overlapped, 2H),

6

-DMSO, supplemental Fig. S12) δ 5.62 (s,

triphenylphosphine converted most of the product

to 17a-OH pregnenolone, as judged by co-

chromatography on UPLC (data not shown).

3

(

1

3

.13 (s, 3H), 1.01-0.78 (overlapped, 2H), 0.57 (s,

H) (57) (see also (58-60)). ESI HRMS: calculated

Synthesis of 17 -OOH progesterone

a

+

for C H O [MH ] m/z 347.2217; found 347.2222

2

1

33

4

Preparation of 17a-OOH progesterone

was done by adapting a previous procedure (41).

Progesterone (1.5 g, 4.0 mmol) was dissolved in

dioxane (150 ml) and H SO (1.5 ml of 2% conc

(

D 1.4 ppm).

Enzymes

Recombinant human P450 17A1 (17), rat

NADPH-P450 reductase (61), zebrafish P450

7A1 and 17A2 (26), and human b (62) and

2

4

H SO in dioxane, v/v) was added followed by

2

4

ethyl orthoformate (4.5 ml, 27 mmol). The mixture

was stirred at room temperature for 1.5 hours,

monitoring development of a (single) product spot

by TLC. The reaction was quenched by addition of

1

5

zebrafish b (26) were expressed in Escherichia

coli and purified as previously reported in detail.ꢀ

5

pyridine (3 ml) and poured into 500 ml of H O.

The product (3-O-ethoxypregnenolone) was

extracted with ethyl acetate, washed once with

2

Reactions

Incubations with P450 17A1

Generally incubations were done at 37 ° C

in a shaking water bath. Time courses were

performed using recombinant P450 17A1 in 100

mM potassium phosphate buffer (pH 7.4) in the

presence of 30 µM L-a-1,2-dilauroyl-sn-glycero-

water and brine, and dried over MgSO ; the

4

solvent was removed using a rotary evaporator.

The crude product was purified by silica gel

chromatography (20% ethyl acetate in hexanes,

v/v). After removal of the solvent, the resulting

product (322 mg, 0.90 mmol) was added to a

mixture of potassium tert-butoxide (1.0 g, 9.0

mmol) in tert-butanol (20 ml). The mixture was

3

-phosphocholine (with or without b ), using

5

general conditions described elsewhere (17, 26,

6). Reconstituted enzyme systems contained

3

P450 17A1 or 17A2, NADPH-P450 reductase, and

^b5 ⁽^w^h^eⁿⁱⁿ^dⁱ^c^a^t^e^d⁾^w^e^r^e^a^d^d^e^dⁱⁿ¹^:²^:¹^m^o^l^a^r

stirred at room temperature while bubbling O gas

2

directly in the solvent. After about 40 min the tert-

butanol began to freeze; 8 ml of tetrahydrofuran

ratios (17, 26). Human b was used with human

5

1

ꢀ

P450 17A structure and functionꢀ

P450 17A1 and zebrafish b₅was used with

zebrafish P450s 17A1 and 17A2. An NADPH-

generating system was used, consisting of 10 mM

column (50 mm ´ 2.1 mm, 1.7 µm) with the

following solvent system—Solvent A: 4.9%

CH CN, 0.1% HCO H, 95% H O; Solvent B: 95%

3

2

+

glucose 6-phosphate, 0.5 mM NADP , and 1 I.U.

CH CN, 0.1% HCO H, 4.9% H O (v/v). Samples

3 2 2

yeast glucose 6-phosphate dehydrogenase/ml (63).

In most of the catalytic assays done with NADPH-

supported systems, the P450 concentration was

were injected with a 20 µl loop and separated with

the following linear gradient for detection of lyase

products: 20% to 80% B (with 17a-OOH

pregnenolone) or 20% to 50% B (all v/v) (with

0

.1-0.5 µM. Typically the reaction time was 1-5

min.

1

7a-OOH progesterone) over 10 minutes at a flow

In the experiments described with a search

-1

rate of 0.2 ml min . Absorbance (diode array, UV,

00-350 nm) was monitored and used for

for conversion of progesterone to 17-O-acetyl

testosterone (Fig. 6), the human P450 17A1

concentration was 1 µM (with 2 µM NADPH-

2

quantitation with external standards. The same

UPLC systems were used to do separations for

P450 reductase, 1 µM b , and 20 or 200 µM

5

mass spectrometry. A₂measurements were used

44

progesterone) and the incubations were run for as

long as 60 min at 37 °C.

4

for quantitation of D steroids.

For the analysis of any 17-O-acetyl

testosterone formed (Fig. 6), the UPLC system

utilized the same column but a gradient of

Alternatively, incubations were initiated

by the addition of 17a-OOH pregnenolone or 17a-

OOH progesterone to a final concentration of 25

µM in (0.5% C H OH, 0.5% (CH ) SO, v/v). No

increasing CH OH in aqueous 0.1% (v/v)

3

2

5

3 2

CF CO H: 0-5 min: 5% CH OH; 5-10 min: 5 to

3

2

3

NADPH-reductase or NADPH was used in these

experiments.

8

0% CH OH; 10-11 min: 80-100% CH OH; hold

3 3

(

all v/v).

Aliquots (500 µl) were removed and

quenched by the addition of 2 ml of CH Cl ,

2

MS and HRMS

3

followed by centrifugation (3 ´ 10 ´ g, 10 min) to

separate the layers. Aliquots (1.7 ml) of the

CH Cl phase were dried and resuspended in 100

LC-MS analyses of 17a-OOH-steroid

transformation was done with samples by UPLC

as described above. A Thermo LTQ-Orbitrap mass

spectrometer was generally used in the FTMS

mode at 7500 resolution. The mass spectrometer

was operated in the electrospray (ESI) positive ion

mode and was tuned with authentic pregnenolone.

The tune settings were as follows: sheath gas flow

rate: 30, aux gas flow rate: 10, sweep gas flow

rate: 0, capillary temperature: 275 °C, source

voltage set to 5 kV, source current: 100 µA,

capillary voltage: 29 V, tube lens: 80 V.

2

µl of CH CN and diluted with 150 µl water for

3

5

HPLC separation. D -Steroids (from pregnenolone

4

series) were oxidized to D -steroids (with

increased UV absorbance) for quantitation (HPLC,

A ) using cholesterol oxidase, as described in

2

44

more detail previously (26, 35).

For steady-state

reconstituted system with NADPH and NADPH-

kinetic

assays

(

P450 reductase), plots of reaction velocity vs.

substrate concentration were fit to hyperbolic plots

to estimate k_c_a_tand K_m(± SD) using Prism

software (GraphPad, La Jolla, CA). The calculated

SD values from Prism were used to estimate the

SD values for the ratio k /K (square root of the

Crystallization of the zebrafish P450 17A1

ETCMM-mutant protein in complex with

abiraterone

cat

m

To generate the P450 complex, the

sum of the squares of SD values for the two

parameters).

inhibitor

abiraterone

(dissolved

in

dimethylsulfoxide) was added to purified zebrafish

P450 17A1-ETCMM mutant protein in a protein

to arbiraterone molar ratio of 1:10. The complex

was concentrated to 30 mg of protein/ml in 50 mM

potassium phosphate buffer (pH 7.4) containing

10% glycerol (v/v), 0.1 mM dithiothreitol, 1 mM

EDTA, 0.004% C12E9 detergent (w/v), and 125

mM NaCl. Prior to setting up crystallization

Chromatography

UPLC, with UV-visible absorbance

detection, was routinely used for measurement of

product formation. Products were generally

separated using a Waters Acquity UPLC system

and an Acquity UPLC-BEH octadecylsilane (C )

1

8

1

2

ꢀ

P450 17A structure and functionꢀ

droplets, subtilisin was added to the complex such

that the protease to P450 17A1 ratio (w/w) was

regions of positive F -F_cdifference Fourier

o

electron density (2.5 σ level) and the refinements

were continued. Water molecules and ions

(chloride and acetate) were added and refined.

Selected refinement parameters are summarized in

Table 4. All structural illustrations were prepared

with the program UCSF Chimera (51).

1

:1000. Crystals were grown using the sitting-drop

vapor diffusion technique by mixing equal

volumes of complex solution and Crystal Screen 1

(

2

Hampton Research, Aliso Viejo, CA) condition

1 (0.2 M magnesium acetate, 0.1 M sodium

cacodylate (pH 6.5), and 30% (v/v) (±)-2-methyl-

,4-pentanediol) and equilibrating the droplets

2

Molecular docking

against 60 μl of reservoir solution at 4 °C. Crystals

of the zebrafish P450 17A1-ETCMM-abiraterone

complex appeared within a few days. One round

of microseeding was performed to optimize the

size of crystals. Wells containing microcrystals

were washed with 10 μl of reservoir buffer, and

Models of complexes between the

zebrafish P450 17A1-ETCMM mutant protein and

17a-OH progesterone, 17a-OH pregnenolone,

1

6,17a-(OH)₂

progesterone,

16,17a-(OH)₂

pregnenolone, and 6b,16,17a-(OH) progesterone

3

(

Fig. 9) were built with Coot (67) and taking into

the mixture was then transferred to

a

account the structures of the complex described in

the present contribution, the complex between

human P450 17A1 and pregnenolone (PDB ID

microcentrifuge tube with 40 μl of mother liquor

and a PTFE Seed Bead (Hampton Research, Aliso

Viejo, CA). After mixing the contents of the tube

with a vortex device and diluting 1:1000 with

mother liquor (v/v), 200 nl of the solution was

mixed with 200 nl of protein for each drop. Larger

crystals of the complex (with typical sizes of 0.2 to

4

NKW) (50), and the complex between human

P450 21A2 and 17a-OH progesterone (PDB ID

VBU) (69). Missing hydroxyl groups were added

5

and the substrate/product molecules oriented in the

active site such that hydroxyl oxygen atoms were

at a distance of between ca. 4 Å (16a-/17a-OH)

and ca. 5 Å (6b-OH) from the Fe(III) atom.

Hydrogen atoms were added automatically and the

initial models then refined using Amber ff14SB in

UCSF Chimera (51), by taking into account steric

and hydrogen bonding interactions and applying a

minimization protocol of 100 steepest descent

steps with a step size of 0.02 Å, followed by 10

conjugate gradient steps with a step size of 0.02 Å.

0

.25 mm) grew from micro-seeded droplets.

Crystals were mounted in nylon loops and flash

frozen in liquid nitrogen.

Structure determination and refinement

X-ray diffraction data were collected on

the 21-ID-F beam line of the Life Sciences

Collaborative Access Team (LS-CAT), located at

Sector 21 of the Advanced Photon Source,

Argonne National Laboratory (Argonne, IL), at a

wavelength of 0.9787 Å and using a Mar225 CCD

detector. Data were processed with the program

xia2/XDS (64) and Pointless/Aimless (64, 65).

Selected crystal data and data collection statistics

are summarized in Table 2. The structure of the

zebrafish P450 17A1-ETCMM mutant in complex

with abiraterone was solved by the molecular

replacement technique with the program Phaser

(

66), using the native zebrafish P450 17A1

structure as the starting model (PDB ID 4R1Z;

protein alone) (33). The N- and C-terminal regions

as well as several loops in the two independent

molecules per asymmetric unit were manually

built into the electron density maps using the

program Coot (67). The structure was refined with

the program Refmac 5 (68) in the CCP4 suite

(

CCP4, 1994, 54289). After about 10 rounds of

refinement, heme and abiraterone were built into

1

3

ꢀ

P450 17A structure and functionꢀ

Acknowledgements: We thank K. Trisler for assistance in preparation of the manuscript.

ꢀ

Conflict of Interest: The authors declare that they have no conflicts of interest with the contents of this

article.

Author contributions: E.G., W.Z., L.L., and T.T.N.P. purified the enzymes, E.G. performed site-directed

mutagenesis, L.L. crystallized the zebrafish P450 17A1-ETCMM mutant protein, Z.W. helped with X-ray

data collection and processing, P.S.P. and M.E. determined the crystal structure, K.M.J. synthesized and

purified the hydroperoxides, F.K.Y., E.G., F.P.G., and M.E. designed the experiments, E.G., W.Z.,

T.T.N.P., and K.M.J. did catalytic assays, K.M.J., T.T.N.P., and W.Z. did mass spectrometry assays, and

F.P.G. and M.E. wrote most of the manuscript. F.P.G. and M.E. revised the manuscript.

ꢀ

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18

P450 17A structure and functionꢀ

FOOTNOTES

S

This paper contains Supporting Information.

This work was supported by National Institutes of Health Grants R01 GM118122 (to F.P.G.), R01

GM103937 (to M.E. and F. P. G.), and T32 ES007028 (E.G.). The authors declare that they have no

conflicts of interest with the contents of this article. The content is solely the responsibility of the authors

and does not necessarily represent the official views of the National Institutes of Health.

1

These authors contributed equally to this work.

2

This paper constitutes part of the requirements to fulfill a Ph.D. thesis at Vanderbilt University. Current

address: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health,

Bethesda, MD 20892.

3

Current address: Department of Drug Metabolism and Pharmacokinetics, Genentech, 1 DNA Way,

South San Francisco, CA.

4

Current address: Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of

Sciences, No. 189 Songling Road, Laoshan District, Qingdao, Shandong, People’s Republic of China.

5

To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of

Medicine, 638B Robinson Research Bldg., 2200 Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-

2

261; Fax: 615-343- 0704; E-mail: f.guengerich@vanderbilt.edu.

Abbreviations: Andro, androstenedione; APCI, atmospheric pressure chemical ionization; DHEA,

dehydroepiandrosterone; ESI, electrospray ionization; HRMS, high resolution mass spectrometry; OH,

hydroxy; -OOH, hydroperoxy; P450, cytochrome P450 (also CYP); P450 17A1-ETCMM, zebrafish P450

1

7A1 containing the mutations D309E, V321T, R369C, L372M, and L407M (supplemental Fig. S1);

P450 17A2-DVRLL, zebrafish P450 17A2 containing the mutations E298D, T310V, C358R, M361L, and

M396L (supplemental Fig. S1); r.m.s.d., root mean square deviation.

ꢀ

19

P450 17A structure and functionꢀ

Table 1.

Progesterone 17a-hydroxylation activities of P450 17A Arg/Cys mutant proteins

Human P450 17A1

Zebrafish P450 17A1

Zebrafish P45017A2

a

b

WT

R358C

WT

R369C

WT

C358R

-

1

k_c_a_t, min

10.2 ± 0.6

4.3 ± 0.4

2.4 ± 0.2

8.3 ± 0.3

3.6 ± 0.5

2.3 ± 0.3

0.8 ± 0.1

0.37 ± 0.01

42 ± 1

23 ± 2

K , µM

0.64 ± 0.06 0.90 ± 0.15 4.4 ± 0.6

1.2 ± 0.2 0.41 ± 0.07 9.4 ± 1.3

7.7 ± 1.3

3.0 ± 0.6

m

-

1

-1

k /K , µM min

cat

m

All rates were measured in the presence of an equimolar concentration of b relative to P450

5

(

human b was used with human P450 17A1 and zebrafish b was used with zebrafish P450s),

5

with a 2-fold molar excess of NADPH-P450 reductase and equimolar concentration of b when

5

indicated.

a

From ref. (36)

b

From study presented in supplemental Table S1.

ꢀ

20

P450 17A structure and functionꢀ

Table 2. Selected crystal data, data collection and refinement parameters for the P450 z17A1-

ETCMM mutant abiraterone complex. ꢀ

Data collection

Wavelength [Å]

0.9787

Space group

P4 22

1

a

Resolution (outer shell) [Å]

51.26 - 3.03 (3.11 - 3.03)

104.92, 235.00; 90

Unit cell constants a=b, c [Å]; a= b=g [°]

Unique reflections

Completeness [%]

I/σ(I)

26,303 (1,896)

99.8 (99.7)

14.0 (2.0)

R-merge

0.119 (0.959)

0.047 (0.371)

7.2 (7.4)

R-pim

Redundancy

Refinement

Phasing method

R-work

Molecular Replacement

b

0.192 (0.301)

b

R-free

0.256 (0.395)

c

Number of amino acids

Number of protein atoms

Number of heme/ligand atoms

935

7,390

86 / 52

2

Average B-factor, protein atoms [Å ]

78.3

2

Average B-factor, heme/ligand atoms [Å ]

58.5 / 60.7

57.8 / 89.6

0.011

2

Average B-factor, water/ions [Å ]

R.m.s.d. bond lengths [Å]

ꢀ

21

P450 17A structure and functionꢀ

R.m.s.d. bond angles [°]

1.6

Ramachandran plot: No of favored / allowed /

outlier residues

8

59 / 59 / 11

6B82

PDB ID code

a

Numbers in parentheses refer to the outer shell.

Outer shell for refinement is 3.108-3.03 Å.

b

c

For two independent molecules per asymmetric unit. Molecule A: residues 39-175 / 180-507.

Molecule B: residues 39-508.

ꢀ

22

P450 17A structure and functionꢀ

Fig. 1

2

1

CH₃

OH

O

18

^C^H3

O

12

20

1

7

11

19

13

1

4

16

2

9

14

15

+ CH CO H

3 2

1

0

8

7

3

O

5

O

6

Progesterone

17 -OH Progesterone

Androstenedione

O

CH₃

OH

O

^C^H3

O

+

CH CO H

3 2

HO

17 -OH Pregnenolone

HO

Pregnenolone

DHEA

Figure 1.

Major reactions catalyzed by P450 17A1 enzymes.

ꢀ

23

P450 17A structure and functionꢀ

Fig. 2

O

OH

A

O

CH3

OH

O

Androstenedione

O

CH₃

O

21

CH3

OH

O

1

7

OH

1

7α-Progesterone

1

6

O

CH3

OH

O

6

OH

O

Algestone

O

Progesterone

CH2OH

OH

O

CH2OH

O

11-Deoxycorticosterone

O

OH

B

11-Deoxycortisol

2

1

O

CH3

OH

CH3

HO

1

7

DHEA

O

1

6

CH3

OH

HO

1

7α-Pregnenolone

Pregnenolone

HO

O

CH2OH

OH

HO

Figure 2.

Oxidations catalyzed by human and zebrafish P450 enzymes (17). Products of the

zebrafish P450s identified in this work include 17a,21-(OH) progesterone (11-deoxycortisol), 16,17a-

2

(

OH)₂progesterone (algestone), 6b,16,17a-(OH)₃progesterone, 17a,21-(OH)₂pregnenolone, and

1

6,17a-(OH) pregnenolone.

2

ꢀ

24

P450 17A structure and functionꢀ

Fig. 3

Progesterone17-hydroxylation

Pregnenolone 17-hydroxylation

1

7OH-Progesterone lyase

1

7OH-Pregnenolone lyase

80

60

40

20

0

4

3

2

1

0

A

B

5

b

LL+b

1

7A1- b1 7A1+

17A2- b1 7A2+

R

17A1-b

17A1+

V

1

7A2- D1 7V AR 2L - LD -b

1

7A1- E1 T7 CA M1 - ME T- bC MM+b

17A1-ETCMM- b1 7A1-ETCMM+b

0.06

0.04

0.02

0.00

0.04

0.03

1

7OH-Progesterone

1

7OH-Pregnenolone

C

D

6

1

2

,16-(OH)₂

6-OH

16-OH

21-OH

1-OH

0

.02

.01

0

0.00

5

b

5

b

5

LL+b

1

7A2-b

17A2+

R

17A2-b

17A2+

R

V

1

7A2-DVRLL-b17A2-D

17A2-DVRLL-b17A2-D

Figure 3.

Summary of steady-state kinetic parameters of zebrafish P450s 17A1 and 17A2 and

the mutants. Parameters are shown for the reactions done in the absence and presence of zebrafish b . No

5

lyase activity was detected for zebrafish P450 17A2 or the mutant derived from it (DVRLL). A, 17a-

hydroxylation with progesterone or pregnenolone as substrate; B, 17a,20-lyase activity with 17a-OH

progesterone or 17a-OH pregnenolone as substrate. C, the substrate was 17a-OH progesterone and the

indicated three minor products were measured; D, the substrate was 17a-OH pregnenolone and the

indicated two minor products were measured. All values are catalytic efficiencies (k /K , ± SD); see

cat

m

supplemental Tables S1, S2 and Figs. S3-S8 for details regarding the substrate concentrations used and

the error analysis for k and K values.

cat

m

ꢀ

25

P450 17A structure and functionꢀ

Fig. 4

Figure 4.

Crystal structure of zebrafish P450 17A1-ETCMM, bound to abiraterone. A, overall

view of the complex with the P450 protein colored in rainbow style, from N terminus (blue) to C terminus

red). Individual a-helices are labeled and every tenth residue is numbered. Heme carbon, nitrogen, and

(

3

+

oxygen atoms are colored in black, blue, and red, respectively, and Fe is shown as an orange sphere.

Carbon and oxygen atoms of the abiraterone inhibitor are colored in magenta and red, respectively. B,

quality of the omit 2F -F Fourier electron density (1.2s level) around abiraterone. C, an overlay of the

o

c

two complexes per crystallographic asymmetric unit reveals highly similar conformations of molecules A

(

beige ribbon) and B (light blue ribbon).

ꢀ

26

P450 17A structure and functionꢀ

Fig. 5

Figure 5.

Environments of the five mutated residues in the structure of the zebrafish P450

1

7A1-abiraterone complexes. A, overlay of wild-type zebrafish P450 17A1 (lilac ribbon, side chains

shown in stick mode) (33) and zebrafish P450 17A1-ETCMM (light blue ribbon, side chains of mutated

residues shown in ball and stick mode), with individual mutants labeled and the closest distance between

3

+

a side chain atom from a mutated residue to the Fe atom, shown in Å. B – E, close-up views: B, D309E;

C, V321T and L372M; D, R369C and L372M; and E, L407M. In panels B – E, side chains of mutated

residues are highlighted in a ball and stick mode. Carbon atoms of side chains are colored lilac (wild type)

and light blue (mutant), and heme and inhibitor carbon atoms in the two structures are colored in an

analogous fashion. Amino acids in the vicinity of mutated residues along with the mutant residue are

labeled, and contacts with distances of < 3.3 Å in the zebrafish P450 17A1-ETCMM mutant complex are

indicated by thin solid lines.

ꢀ

27

P450 17A structure and functionꢀ

Fig. 6

O

H–O

O

O O-Fe

FeO ^–

2

[

FeO]³⁺

O

H–O

O

O O-Fe

OH

_F_e_O–

2

O–H

OH

+

CH CO H

3 2

Figure 6.

Baeyer-Villiger reaction proposed for conversion of progesterone to testosterone 17-

O-acetate (38, 39). This is a modification of a ferric peroxide mechanism (12).

Fig. 7

O

H

[

O

Fe]³⁺

HO

(

Compound I)

O

a

O O–Fe

O

+

CH CO H

3 2

O

b

O

O O–H

Fe³⁺

O

1

7 -OOH Pregnenolone

7 -OOH Progesterone

Figure 7.

Hypothesized conversion of 17

a

-OOH steroids to lyase products (b) and proposed

relationship to normal catalysis (a) (32).

ꢀ

28

P450 17A structure and functionꢀ

Fig. 8

2

1

5

.0 10⁷

15

+

P450 17A1

C

A

P450 17A1 + b₅

.5 10⁷

1

0

5

0

DHEA

.0 10⁷

1

7αOOH Preg

6

.0 10 17αOH preg

-

P450 17A1

0

2

4

6

8

10

0

.0

5

.25

5.50

5.75

6.00

6.25

6.50

3

2

1

000000

0

1

5

B

D

+

P450 17A1

0

5

0

1

7αOOH control 150 s

7αOOH + P450 150 s

-

P450 17A1

8 10

1

0

2

4

6

5

.25

5.50

5.75

6.00

6.25

6.50

Time, min

t_R, min

Figure 8. Conversion of 17

a

-OOH pregnenolone to DHEA by human P450 17A1. A, HPLC of

standard 17a-OOH pregnenolone, 17a-OH pregnenolone, and DHEA, showing t_Rfor elution. B,

chromatograms of reaction of 25 µM 17a-OOH pregnenolone without (green) or with (blue) 60 nM

human P450 17A1 after 150 seconds. Absorbance (relative units) was integrated over the UV range (200-

3

50 nm). See experimental Procedures for other details regarding chromatography. C, rate of conversion

of 17a-OOH pregnenolone to DHEA, ± human b . D, rate of conversion of 17a-OOH progesterone to

5

Andro. The points indicate single assays done at each time.

ꢀ

29

P450 17A structure and functionꢀ

Fig. 9

ꢀ

30

Article Doi

Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes

DOI: 10.1074/jbc.RA117.000504

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.RA117.000504

Full text of DOI:10.1074/jbc.RA117.000504

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