(Received for publication, January 4, 1996; and in revised form, February 19, 1996)
From the
Human P450 2C19 is selective for 4`-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the
structurally homologous P450 2C9 has low activity toward these
substrates. To identify the critical amino acids that determine the
specificity of human P450 2C19, we constructed chimeras of P450 2C9
replacing various proposed substrate binding sites (SRS) with those of
P450 2C19 and then replaced individual residues of P450 2C9 by
site-directed mutagenesis. The 339 NH-terminal amino acid
residues (SRS-1-SRS-4) and amino acids 160-383
(SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase
activity to P450 2C9. In contrast, the COOH terminus of P450 2C19
(residues 340-490 including SRS-5 and SRS-6), residues
228-339 (SRS-3 and SRS-4) and residues 292-383 (part of
SRS-4 and SRS-5) conferred only modest increases in activity. A single
mutation Ile
His increased omeprazole 5-hydroxylase
to
51% of that of P450 2C19. A chimera spanning residues
160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase
activity which was dramatically enhanced by the mutation Ile
His. A combination of two mutations, Ile
His and Ser
Pro, converted P450 2C9
to an enzyme with a turnover number for omeprazole 5-hydroxylation,
which resembled that of P450 2C19. Mutation of Pro
Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids
220 and 221 are in the F-G loop and outside any known SRS. Mutation of
these three amino acids did not confer significant S-mephenytoin 4`-hydroxylase activity to P450 2C9, although
chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450
2C19 exhibited activity toward this substrate. Our results thus
indicate that amino acids 99, 220, and 221 are key residues that
determine the specificity of P450 2C19 for omeprazole.
The P450 ()cytochromes represent a ubiquitous
superfamily of monooxygenases, which metabolize a vast array of
endogenous and exogenous
substrates(1, 2, 3) . Multiple P450 enzymes
appear to have arisen from a single ancestral gene by duplication and
diverged by mutation and gene conversion to produce families of
structurally related enzymes with overlapping but often distinct
substrate specificities. The regio- and stereoselectivity of specific
enzymes for particular substrates appears to be encoded in certain
defined regions of the primary sequence. Considerable progress has been
made in recent years in elucidating the structural determinants of
substrate specificity(4) . In some cases, substrate specificity
between highly related members of the same subfamily has been shown to
be defined by a few critical residues or even a single amino
acid(5) .
P450 2C19 is a member of the human CYP2C subfamily, which includes four structurally related enzymes(6) . P450 2C9 and 2C19 are the most highly conserved of these forms, showing 91% structural identity, but have very distinctive substrate specificities. For example P450 2C19, which is polymorphic in man, is the principal enzyme responsible for the stereoselective 4`-hydroxylation of S-mephenytoin (7) and is highly selective for the 5-hydroxylation of the popular anti-ulcer drug omeprazole(46) . The structurally related P450 2C9 exhibits little activity toward either of these substrates, but exhibits a high turnover number for hydroxylation of tolbutamide and phenytoin and specifically 7-hydroxylates S-warfarin(6) . The present studies were designed to elucidate the key amino acids that determine the marked specificity of P450 2C19 for omeprazole and S-mephenytoin.
Since none of the mammalian P450 enzymes
have been crystallized, the experimentally derived three-dimensional
crystal structures of the bacterial enzymes, P450cam, P450BM-3, and
P450terp, have served as models for predicting their structure and
function. The current paradigm for such predictions has focused on
alignment-based sequence similarities, - and
-secondary
structure, hydropathy indices, computerized modeling based on the
crystal structures of the bacterial P450 enzymes and protein
engineering using chimeric constructs and site-directed
mutagenesis(8, 9, 10, 11, 12) .
Although the amino acid sequences of the soluble bacterial P450 differ
considerably from those of the membrane-bound mammalian P450 enzymes,
these methods have proven resourceful in identifying regions that
determine the substrate specificities of the mammalian proteins.
Gotoh (13) predicted six potential substrate recognition sites (SRS) in the mammalian family 2 P450 enzymes based on an alignment with bacterial P450cam whose substrate-binding residues have been identified by x-ray crystallography and analysis of mutations that altered substrate specificity in experimental studies of the P450 2 subfamily. For example, site-directed mutagenesis studies have shown that the substrate and regiospecificity of the 2C subfamily of cytochrome P450 can be altered by substitution of critical amino acid residues at positions 112 to 115, 301, 359, 364(14, 15, 16, 17, 18, 19) . Replacement of residues 117, 209, 365, and 481 in the 2A subfamily (5, 20, 21, 22, 23) as well as residues 114, 206, 302, 363, 367, and 478 (24, 25, 26, 27, 28) in the 2B subfamily and residue 380 (29) in 2D1 have also been documented to alter substrate or regiospecificity.
To identify key amino acid residues responsible for the marked specificity of human P450 2C19 for two key substrates, omeprazole and S-mephenytoin, we constructed a total of 24 chimeras and mutant enzymes from the structurally related P450 2C9. Constructs were expressed in a yeast cDNA expression system. Initial chimeras replaced regions of P450 2C9 with similar segments of P450 2C19, using restriction enzymes sites located outside the six putative substrate binding domains proposed by Gotoh(13) . Subsequently, we used primary sequence alignment and comparative analysis of amino acid variations within the appropriate segments of the human P450 2C proteins to identify residues that might play a role in the substrate specificity of P450 2C19. Site-directed mutagenesis was then used to identify key residues responsible for the substrate specificity of P450 2C19.
Figure 1:
Schematic
representation of the chimeric constructs (A) and
site-directed mutants (B) of P450 2C9 and P450 2C19. A, the restriction enzyme sites utilized for chimeras and
putative SRS (numbered 1-6) are indicated at the
appropriate sites of P450 2C9 (top). Solid thin lines of the chimeras represent segments of P450 2C19, while bars represent the segments of P450 2C9. The site of an I99H mutation
in a 2C9/2C19 chimera is indicated by a diamond. B, site-directed mutants of P450 2C9. The
amino acids shown above the construct represent those of P450 2C9 which
were mutated to the corresponding residues of P450 2C19 shown below the
construct. The region 198-240 between SRS-2 and SRS-3 is expanded
below. Residues that affected omeprazole hydroxylase activity are
represented by diamonds.
Figure 2: Omeprazole 5-hydroxylase activity of chimeras of P450 2C9 and P450 2C19 expressed in yeast. Construction of chimeras is shown in Fig. 1. The omeprazole hydroxylase activity was measured as described under ``Experimental Procedures.''
Figure 3: Omeprazole 5-hydroxylase activities of site-directed mutants of P450 2C9. Omeprazole hydroxylase activity is given as the means ± S.E. The single mutations are numbered m1-m5, with amino acid substitutions indicated in parentheses, and m134, m145, m1345, and m1245 represent the corresponding multiple mutants. Three other mutant enzymes, m2 (S451F), m34, and m45 (not shown), did not affect omeprazole hydroxylase activity.
Figure 4:
Model showing omeprazole docked in the
substrate-binding pocket of P450cam. Thr is the highly
conserved Thr in the I helix. The heme is shown in red, amino
acid residues in blue, and substrate in black.
Figure 5:
Analysis of the contribution of His and region 160-227 of 2C19 to omeprazole 5-hydroxylase
activity using chimeras and site-directed mutants. The construction of
the 2C9/2C19
chimera is shown in Fig. 1.
A second 2C9/2C19
chimera contained the
Ile
His mutation. Individual amino acids in the
160-227 region were mutated in P450 2C9 in combination with the
Ile
His mutation as shown in Fig. 1B. Values represent means ±
S.E.
We next sequentially modified
amino acids in this region to those of the corresponding residues in
P450 2C19 as shown in Fig. 1B. Six amino acids of P450
2C19 (three conservative and three nonconservative changes) differ from
those of P450 2C9 between amino acids 160 and 227, with five of these
mapping within 20 tandem amino acid residues of the carboxyl end of
SRS-2. Mutations Lys
Arg (K206R), Leu
Val (L208V), Pro
Thr (P221T), and
Ser
Thr in combination with Ile
His did not increase omeprazole 5`-hydroxylase activity
over that of I99H alone (Fig. 5), indicating that these residues
are not the critical amino acids in this region. However, mutation of
the polar serine at position 220 to a nonpolar hydrophobic proline
residue (Ser
Pro) increased activity to
90%
of that of P450 2C19. When a second nonconservative mutation was made
in the tandem residue at position 221 (P221T) in conjunction with S220P
and I99H, the omeprazole hydroxylase activity of the triple mutant
(I99H/S220P/P221T) was equivalent to that of P450 2C19. Sequence
alignment indicates that residues 220 and 221 reside midway between the
COOH-terminal end of the F-helix and the NH
-terminal end of
G-helix in the interhelical loop, outside of any known substrate
binding sites. This region corresponds to the F-G helical turn. This
region is extremely variable in length in the P450 superfamily, and
bacterial P450cam contains no amino acids corresponding to Pro
or Thr
of P450 2C19. The three-dimensional model of
P450 2B1 would predict that these amino acids are far from the
substrate-binding pocket; however, it has been suggested that this
region may form a flexible lid over the substrate-binding pocket and
thus form part of the substrate access
channel(8, 11) . Proline residues give loops greater
flexibility. Therefore, the introduction of Pro
may
refold the F-G loop and change accessibility to the active site or
bring these residues closer to the substrate pocket. Interestingly, Uno
and Imai (44) reported that residues 210-262, which
contain part of the same domain, cooperate with amino acids
90-125 to confer substrate specificity to laurate
(
-1)-hydroxylase P450 2C2. Several other investigators have also
identified important key residues in or near this domain, including
residue 209 of mouse 2A5 as well as 206 and 209 of rat
2B1(5, 20, 22, 26, 45) .
The present results clearly show that residues 220 and 221, which are
in the F-G loop of human P450 2C19 and outside the SRS proposed by
Gotoh(13) , are critical determinants of omeprazole hydroxylase
activity.
Figure 6: S-Mephenytoin 4`-hydroxylase activity of chimeric and mutant CYP2C9 proteins expressed in yeast. Chimeras and mutants are identical to those shown in Fig. 2and Fig. 5. None of the other mutants shown in Fig. 1B, Fig. 3, and Fig. 4increased mephenytoin hydroxylase activity (data not shown).