Identification of phenylalanine 3-hydroxylase for meta -tyrosine biosynthesis

Article abstract of DOI:10.1021/bi200733c

Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.

Full text of DOI:10.1021/bi200733c

RAPID REPORT
pubs.acs.org/biochemistry
Identification of Phenylalanine 3-Hydroxylase for
meta-Tyrosine Biosynthesis
†
,‡
†
,†
Wenjun Zhang, Brian D. Ames, and Christopher T. Walsh*
†
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,
United States
‡
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States
S Supporting Information
b
ABSTRACT: Phenylalanine hydroxylase (PheH) is an
iron(II)-dependent enzyme that catalyzes the hydroxylation
of aromatic amino acid L-phenylalanine (L-Phe) to L-tyr-
osine (L-Tyr). The enzymatic modification has been demon-
strated to be highly regiospecific, forming proteinogenic
para-Tyr (p-Tyr) exclusively. Here we biochemically char-
acterized the first example of a phenylalanine 3-hydroxylase
Figure 1. Examples of m-Tyr (highlighted in red) containing bacterial
secondary metabolites.
(
Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr)
from Phe. Subsequent mutagenesis studies revealed that two
residues in the active site of Phe3H (Cys187 and Thr202)
contribute to C-3 rather than C-4 hydroxylation of the
phenyl ring. This work sets the stage for the mechanistic and
structural study of regiospecific control of the substrate
hydroxylation by PheH.
1
6,17
phenylalanine hydroxylase homologue PacX.
A close protein
homologue SfaA (49% identical to PacX) has recently been
identified in the SFA biosynthetic gene cluster, and inactivation
of SfaA abolished SFA production, which could be restored by
feeding of m-Tyr. This finding strongly indicated that these
phenylalanine hydroxylases could act on Phe to introduce a
hydroxyl moiety at the meta position. In parallel with the sfaA
knockout studies and to probe the regiospecific control mecha-
nism of PheH, we have purified and characterized PacX from the
pacidamycin producer Streptomyces coeruleoribudus.
15
henylalanine hydroxylase (PheH) is a member of the highly
homologous family of aromatic amino acid hydroxylases
P
that also includes tyrosine hydroxylase (TyrH) and tryptophan
1
,2
The catalytic core of PacX more closely resembles that of the
human Phe4H (31% identical) than that of the bacterial Phe4H
from Chromobacterium violaceum (23% identical), yet PacX lacks
the N-terminal regulatory domain and the C-terminal tetramer-
hydroxylase. They are iron(II)-dependent enzymes that typi-
cally utilize molecular oxygen and tetrahydrobiopterin (BH ) as
4
cosubstrates. In accord with their essential functions in human
aromatic amino acid metabolism, this family of enzymes has been
1
8,19
3
ization domain found in human Phe4H
(Figure S1 of the
studied extensively. All PheH enzymes characterized to date are
Supporting Information). PacX was cloned as an N-terminal six-
His-tagged protein, expressed, and purified from Escherichia coli
with a yield of 6.7 mg/L (Figure S2 of the Supporting In-
formation). To directly visualize the formation of Tyr, an HPLC-
based assay was performed using both p-Tyr and m-Tyr as
standards. The purified enzyme was mixed with L-Phe and
phenylalanine 4-hydroxylases (Phe4Hs) yielding the 4-hydroxy-
phenylalanine regioisomer (p-Tyr) as the product. TyrH con-
ducts a hydroxylation of p-Tyr to yield L-DOPA; it also slowly
generates p-Tyr and m-Tyr (25:1 ratio) when presented with
4
the alternate substrate L-Phe. Despite several available crystal
5À9
structures of PheH,
the mechanism of regiospecific hydro-
6
-methyltetrahydropterin (6-MePH ) in sodium phosphate buf-
4
xylation of Phe to form p-Tyr is poorly understood. No enzyme
has been characterized to be dedicated in the catalysis of m-Tyr
formation.
fer, and the mixture was incubated at room temperature, the
reaction quenched with 10% trichloroacetic acid (TCA), and the
mixture subjected to HPLC analysis (Figure 2). m-Tyr was
detected as the product, confirmed by its UV profile, HRMS,
and compared to the standard (Figure S3 of the Supporting
Information). Only trace amounts of p-Tyr were formed from
the reaction mixture (<3% compared to m-Tyr). Control experi-
ments demonstrated that both substrates L-Phe and 6-MePH4
were essential for m-Tyr formation. In addition to L-Phe, the
substrate specificity of PacX toward D-Phe, L-Tyr, and L-Trp was
m-Tyr has been found in humans that could be misincorpo-
10À12
rated into proteins through phenylalanine-tRNA synthetase.
It
is presumably formed from phenylalanine by hydroxyl radicals. In
addition, m-Tyr has been identified as a natural herbicidal
compound produced by fine fescue grasses to displace neighbor-
ing plants; thus, it might be potentially useful in biorational weed
1
3
control. Several bacterial secondary metabolites also contain
m-Tyr as one of the building blocks, including uridyl peptide anti-
biotics such as pacidamycins and a potent cyclophilin inhibitor
14,15
sanglifehrin A (SFA) (Figure 1).
Our recent characterization
Received: May 10, 2011
of the biosynthetic gene cluster for pacidamycins indicated that
Revised:
May 26, 2011
m-Tyr could be formed from Phe, through the function of a
Published: May 26, 2011
r 2011 American Chemical Society
5401
dx.doi.org/10.1021/bi200733c
|Biochemistry 2011, 50, 5401–5403

Biochemistry
RAPID REPORT
Figure 3. Mutagenesis studies of the regiospecific hydroxylation con-
trol of PacX. (a) Fluorescence chromatograms showing production of
m-Tyr and p-Tyr catalyzed by wild-type PacX and its mutants. (b)
Three-dimensional structural model of PacX active site (Phe3H). The
structure of PacX was modeled on the basis of Phe4H (Protein Data
2
3
Bank entry 1KW0) using the on-line program SWISS-MODEL. The
five residues that form the hydrophobic cage around the aromatic side
chain of L-Phe are underlined.
Figure 2. Characterization of PacX as a phenylalanine 3-hydroxylase.
Thr202 (Figure 3), which are conserved among putative Phe3Hs
with the minor exception that Thr202 is Ser in SfaA. Compared
to those of Phe4H and TyrH, three of these five residues are
highly conserved, while Cys187 and Thr202 are replaced by Phe
and Gly, respectively (Figures S1 and S5 of the Supporting Informa-
tion). Thus, we constructed the PacX C187F and T202G single
mutants and the PacX C187F/T202G double mutant to examine
their effects on the activity of PacX in forming m-Tyr versus p-Tyr.
All three mutants were purified from E. coli with decreased yields
of 0.4, 0.5, and 3.9 mg/L, respectively (Figure S2 of the Support-
ing Information). The overall hydroxylation rate was comparable
for the T202G single mutant, the C187F/T202G double mutant,
and the wild-type enzyme, while it was reduced to <4% of the rate
of the wild type for the C187F single mutant (Figure 3a). We
suspect that the inactivity is due to the steric effects of the bulky
side chain of Phe187 in the presence of Thr202, leading to the
possible rearrangement of the active site architecture. The ratio
of product p-Tyr to m-Tyr was changed to ∼2:1 for the T202G
mutant, which represents a 60-fold increase in the preference for
p-Tyr over m-Tyr formation. A more dramatic change in the
product profile was found in the C187F/T202G double mutant
reaction, where only <8% of m-Tyr compared to p-Tyr was
formed (Figure 3a). These results demonstrated that both residues
Cys187 and Thr202 are critical in controlling the regiospecific
hydroxylation of the phenyl ring. It is likely that the two residues
work in concert to clear possible hindrance at the meta position
and to promote hydroxylation by hydrogen bonding. Elucidation
of the exact mechanism will require future structural studies of
Phe/m-Tyr-bound Phe3H.
(a) Scheme of PheH-catalyzed reactions. (b) Fluorescence chromato-
grams (λex = 270 nm, and λem = 310 nm) showing PacX-catalyzed
production of m-Tyr with various control reactions omitting one of the
components indicated. (c) Determination of PacX kinetic parameters by
HPLC assays monitoring the formation of m-Tyr. Parameters were
determined for L-Phe (top) by fixing the 6-MePH
.5 mM, while parameters were determined for 6-MePH
fixing the L-Phe concentration at 5 mM.
4
concentration at
0
4
(bottom) by
tested: PacX was not able to hydroxylate any of these substrates.
The addition of ferrous ammonium sulfate to the reaction mixture
increased the product yield more than 3-fold, strongly indicating
that PacX is an Fe-dependent phenylalanine hydroxylase. DTT
was also found to increase the product yield more than 72%,
20
consistent with a previous report. The effect of pH on PacX
was tested using phosphate buffer at pH values ranging from 5.8
to 7.8 (Figure S4 of the Supporting Information). The activity
of PacX was maximal at pH 6.0, and the enzyme tended to
precipitate gradually below pH 6.5. We thus used a pH value of
6
.2 throughout the studies.
The kinetic parameters of PacX were determined using HPLC
assays monitoring the formation of m-Tyr (Figure 2c). The Km
values of the enzyme were determined to be 26 μM for 6-MePH4
and 1.1 mM for L-Phe, which were comparable to reported values
9
,20À22
À1
for Phe4H originating from different organisms.
The
turnover number was determined to be ∼1.3 min , which
was more than 100-fold lower than those of typical Phe4H. This
low kcat value could reflect a low fraction of properly folded
enzyme from heterologous expression, or more likely, it could be
representative of low kcat values seen with other enzymes in
secondary metabolic pathways. In particular, it may be one way of
reducing the flux of the key metabolite L-Phe to nonproteino-
genic m-Tyr rather than proteinogenic p-Tyr.
In summary, we have characterized in vitro for the first time a
phenylalanine 3-hydroxylase that catalyzes the synthesis of m-Tyr
from L-Phe. The reaction presumably proceeds through a
consensus PheH mechanism in which a high-spin Fe(IV)dO
species is the immediate donor of oxygen to the substrate phenyl-
alanine bound in the active site. Subsequent electrophilic aro-
matic substitution generates a cationic diene species that under-
goes a low-energy hydride shift forming the cyclohexadienone,
The enzymatic control of regiospecific hydroxylation of L-Phe
was probed by site-directed mutagenesis. The residues predicted
to make up the hydrophobic cage surrounding the aromatic side
chain of L-Phe in PacX are Trp182, Cys187, Pro136, His140, and
5
402
dx.doi.org/10.1021/bi200733c |Biochemistry 2011, 50, 5401–5403

Biochemistry
RAPID REPORT
2
,24,25
followed by aromatization to yield the product phenol
(
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Figure S6 of the Supporting Information). We furthermore
(
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Kelleher, N. L., and Walsh, C. T. (2011) J. Am. Chem. Soc.
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18) Kobe, B., Jennings, I. G., House, C. M., Michell, B. J., Goodwill,
K. E., Santarsiero, B. D., Stevens, R. C., Cotton, R. G., and Kemp, B. E.
identified a role for residues Cys187 and Thr202 in determining
the regiospecific hydroxylation of the L-Phe substrate. Phe3H
presumably evolved from ancient Phe4H, with the first mutation
of Phe to Cys that would have been a one-base event. This is also
indicated by the inactivity of the C187F mutant of PacX. The
second mutation of Gly to Thr/Ser further increased the
selectivity for the formation of m-Tyr over p-Tyr. It would be
interesting to test if Phe4H could be converted to Phe3H by
mutating the corresponding residues.
1
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ASSOCIATED CONTENT
(
Biochemistry 39, 9652–9661.
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S
Supporting Information. Experimental procedures, se-
b
quence alignment of PheHs and TyrHs, SDSÀPAGE analysis of
purified proteins, UV and HRMS characterization of m-Tyr,
effect of pH on PacX activity, modeled three-dimensional
structure of PacX, comparison of the active site of PacX to
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’
AUTHOR INFORMATION
Corresponding Author
*
Phone: (617) 432-0930. Fax: (617) 432-0556. E-mail: christo-
pher_walsh@hms.harvard.edu.
Funding Sources
This work was supported by National Institutes of Health (NIH)
Grant GM49338 (C.T.W.) and NIH Grant F32GM090475 (B.
D.A.).
’
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dx.doi.org/10.1021/bi200733c |Biochemistry 2011, 50, 5401–5403