Stereochemistry and mechanism of a microbial phenylalanine aminomutase

Article abstract of DOI:10.1021/ja2030728

The stereochemistry of a phenylalanine aminomutase (PAM) on the andrimid biosynthetic pathway in Pantoea agglomerans (Pa) is reported. PaPAM is a member of the 4-methylidene-1H-imidazol-5(4H)-one (MIO)-dependent family of catalysts and isomerizes (2S)-α-phenylalanine to (3S)-β-phenylalanine, which is the enantiomer of the product made by the mechanistically similar aminomutase TcPAM from Taxus plants. The NH₂ and pro-(3S) hydrogen groups at C_α and C_β, respectively, of the substrate are removed and interchanged completely intramolecularly with inversion of configuration at the migration centers to form β-phenylalanine. This is a contrast to the retention of configuration mechanism followed by TcPAM.

Full text of DOI:10.1021/ja2030728

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pubs.acs.org/JACS
Stereochemistry and Mechanism of a Microbial Phenylalanine
Aminomutase
Nishanka Dilini Ratnayake,† Udayanga Wanninayake,† James H. Geiger,† and Kevin D. Walker*,†,‡
†Department of Chemistry and ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing,
Michigan 48824, United States
S Supporting Information
b
Scheme 1. Biosynthetic Pathway to Andrimid
ABSTRACT: The stereochemistry of a phenylalanine
aminomutase (PAM) on the andrimid biosynthetic pathway
in Pantoea agglomerans (Pa) is reported. PaPAM is a member of
the 4-methylidene-1H-imidazol-5(4H)-one (MIO)-depen-
dent family of catalysts and isomerizes (2S)-R-phenylala-
nine to (3S)-β-phenylalanine, which is the enantiomer of
the product made by the mechanistically similar aminomu-
tase TcPAM from Taxus plants. The NH2 and pro-(3S)
hydrogen groups at CR and Cβ, respectively, of the substrate
are removed and interchanged completely intramolecularly
with inversion of configuration at the migration centers to
form β-phenylalanine. This is a contrast to the retention of
β-C-2H6]-(E)-cinnamate (1) and [2-15N]-(2S)-R-phenylalanine
configuration mechanism followed by TcPAM.
(2) (each 98þ% enriched), a mixture of isotopomers unlabeled
(4) and [U-13C,2-15N]-(2S)-R-phenylalanine (5) (98þ% en-
riched), racemate [ring,3-2H6]-(2R,3S)/(2S,3R)-R-phenylala-
nine (8/9) (98þ% enriched), and [ring,2,3-2H7]-(2S,3S)-R-
antoea agglomerans (Pa) bacteria produce the antibiotic
phenylalanine (11) (90% ee, 98þ% enriched) were separately
P
andrimid, a polyketide/non-ribosomal peptide that inhibits
incubated with the aminomutase. The resulting β-phenylalanine
isotopomers were derivatized to their N-benzoyl methyl esters
and analyzed by GC/EI-MS; derivatized unlabeled β-phenylala-
nine was identically analyzed.
the bacterial acetyl coenzyme A carboxylase. One of the building
blocks used during andrimid assembly is (3S)-β-phenylalanine,
derived by isomerization of (2S)-R-phenylalanine via the AdmH
catalytic domain, that functions as a phenylalanine aminomutase
(PAM)1 (designated herein as PaPAM). The primary amino acid
sequence of PaPAM is homologous to a class I lyase-like family of
Based on the reaction of the homologous PAM from Taxus
plants, (E)-cinnamate is a proposed intermediate of the forward
reaction during isomerization of R- to β-phenylalanine and is
reported as a substrate (at pH 10.5 in 6 M NH4þ salt) used to
make a mixture of R- and β-phenylalanines.8 Thus, to evaluate
whether PaPAM could transfer the amino group from phenyla-
lanine to an exogenous (E)-cinnamate, a mixture of 1 and 2 was
incubated with PaPAM. The reactions were quenched, and the
amino acids were derivatized and analyzed by GC/EI-MS.
Analysis of the fragment ions suggested that the β-phenylalanine
(>99%) was 15N-enriched (3) with no unlabeled isotopomer
present and was not derived from [ring,β-C-2H6]-(E)-cinnamate
(Table 1A). The mode of intramolecular exchange was further
confirmed by incubating the aminomutase with a mixture of 4A
and 5. The β-amino acid derivatives were analyzed as before, and
the 14N and 15N of the β-amino acid products (6A and 7,
respectively) remained coupled to the carbon scaffold of the
corresponding R-amino acid substrate (Table 1B).
catalysts (comprised of ammonia lyases2ꢀ4 and aminomutases1,5ꢀ7
)
that contain a signature 4-methylidene-1H-imidazol-5(4H)-one
(MIO) prosthesis. The biosynthetic pathway to andrimid con-
tinues through the AdmJ catalytic domain that adenylates (3S)-
β-phenylalanine and then transfers the activated intermediate to
a free-standing thiolation domain, AdmI. Subsequent octatrie-
noylation, amidation, and ketonization of the β-amino acid by
other enzymatic domains on the assembly line produce the final
product1 (Scheme 1). Herein, we report on the characteristics of
the mechanism and stereochemistry of the PaPAM reaction.
AdmH was cloned and expressed as an N-terminal His6 fusion
from the pET-24b(þ) vector in Escherichia coli (BL21). Activity
assays with expressed PaPAM and (2S)-R-phenylalanine con-
firmed the product as (3S)-β-phenylalanine, after the amino
acids in the reaction mixture were converted to their N-(1(S)-
camphanoyl) methyl esters. The derivatized β-amino acid was
identified by GC/EI-MS analysis to have a retention time
identical to that of authentic N-[(10S)-camphanoyl]-(3S)-β-ph-
enylalanine methyl ester and not the 10S,3R-isomer.
An R-phenylalanine racemate contained the nonproductive
(2R)-enantiomer 8 and the productive (2S)-enantiomer 9; the
reaction stereospecificity was assessed previously by incubating
To assess the mechanism and cryptic stereochemistry of the
proton transfer of the PaPAM reaction, a mixture of [ring,
Received: April 4, 2011
Published: May 11, 2011
r
2011 American Chemical Society
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Table 1. β-Phenylalanine Isotopes Derived from PaPAM
Catalysis and Various Labeled R-Phenylalanines
Scheme 2. Reaction Catalyzed by PaPAM Proceeds with
Inversion of Configuration
(CβD) and 2.83 (CRD) were equal, suggesting that no hydrogen
exchange occurs with the buffer protons during the isomeriza-
tion, as shown earlier.1,9 This contrasts the significant hydrogen
exchange (∼60%) observed over 1 h with another MIO-depen-
dent aminomutase from Taxus plants (TcPAM)5 and suggests
that the PaPAM active site excludes water more effectively. In
addition, the 2H NMR data strongly support a mechanism where
PaPAM shuttles the pro-(3S) hydrogen from C3 of (2S)-R-
phenylalanine to C2 of the β-isomer product with inversion of
configuration, while the amino group reattaches at C3, also with
stereochemical inversion (Scheme 2). This is distinctly different
than the retention of configuration mechanism of the TcPAM
reaction.10
As a member of the MIO-based aminomutases, PaPAM likely
proceeds through a stepwise mechanism where the migratory
hydrogen and amino group are eliminated heterolytically from
the substrate and held by the enzyme.11 An ensuing trans-acrylate
intermediate is proposed to provide the carbon skeleton upon
which the amino group and hydrogen rebound with inversion of
configuration.
To invoke the proper stereochemistry, PaPAM likely removes
and reattaches the amino and hydrogen groups to the same face
of the cinnamate intermediate (16A) from which they originated
(Scheme 3), whereas TcPAM is proposed to remove and reattach
the amino and hydrogen groups to the opposite face from which
they originated (cf. 18A), possibly via rotation of the intermedi-
ate in the active site10 (Scheme 3). More importantly, the
cinnamate intermediate on both pathways (cf. Scheme 3) is
retained in the active site throughout the course of each isome-
rization reaction, as evidenced herein by incubating PaPAM with
1 and 2 to make 3 exclusively. A similar assay with TcPAM in a
previous study showed essentially the same results.10
PaPAM (2 mg) separately with unlabeled (2S)- and (2R)-R-
phenylalanine (data not shown). The β-amino acid derivatives
analyzed by GC/EI-MS indicated that the deuterium at the
pro-(3R) position of the substrate was retained in the (3R)-β-
phenylalanine product (10) (Table 1C). In contrast, the same
analysis of the derivatized β-phenylalanine isotopomer isolated
after incubation of PaPAM with 11 indicated that no deuterium
remained at C3 of the product 12 (Table 1D). All of the
deuterium at the pro-(3S) migrated to C2, suggesting that
this migration was completely intramolecular without exchange
with solvent protons. This latter observation supported an
earlier study that showed all three of the deuteriums of
[2H8]-R-phenylalanine, after incubation with PaPAM, were
completely retained in the β-amino acid.9
Evidence to support the proposed pathways for the PaPAM
and TcPAM reactions was provided by incubating 20-methyl-
(2S)-R-phenylalanine (15B, 1 mM) at 31 °C for 1 h separately
with each enzyme. The reactions were acidified, and the cinnamic
acid analogues were separately extracted into ethyl acetate and
converted to their methyl esters. The remaining aqueous frac-
tions were basified, and the amino acids were derivatized to their
ethyl carbamates with ethyl chloroformate, acidified, extracted
into ethyl acetate, and finally methyl esterified. An aliquot of each
extract was separately analyzed by GC/EI-MS. The distribution
of 20-methyl-β-phenylalanine and 20-methyl-(E)-cinnamate made
from 20-methyl-(2S)-R-phenylalanine by PaPAM catalysis was
98:2, while a reciprocal distribution (∼1:99) was observed for
TcPAM catalysis. Comparison of the kinetic parameters for
The stereochemistry of the hydrogen rebound during the
aminomutase reaction was evaluated by incubating [3,3-2H2]-R-
phenylalanine (13) with PaPAM for 2 h. Thereafter, the amino acid
isotopomers (including 14, Table 1E) were derivatized as their
N-acetyl methyl esters and dissolved in CHCl3 for 2H NMR analysis.
2
The H NMR profile of the derivatized [2H]-labeled amino
acid mixture contained peaks at δ 5.40 (NCβD) and 2.83
(HCRD), which were identical to those of the authentic racemate
[2,3-2H2]-N-acetyl-(2S,3R)/(2R,3S)-β-phenylalanine methyl
ester.5 These NMR data coupled with the known (3S)-stereo-
chemistry1 (also found herein) of the biosynthetically derived
[2H]-β-phenylalanine product established the biosynthetic pro-
duct as the (2R,3S)-enantiomer. Further, the integrals of the peak
areas (set to 1.0 deuterium) for the resonance signals at δ 5.40
PaPAM (kcat = 0.061 sꢀ1, β-amino acid production; KM
=
0.05 mM) and TcPAM (kcat = 0.002 sꢀ1, cinnamate production;
KM = 0.01 mM) with 20-methyl-(2S)-R-phenylalanine showed
that the catalytic efficiency (kcat/KM = 1.2 sꢀ1 mMꢀ1) of PaPAM
was 6-fold greater than the efficiency of TcPAM, due largely to
the superior kcat.
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Scheme 3. Route A: Proposed Course of PaPAM Reaction from (2S)-R-Phenylalanine to (3S)-β-Phenylalanine; Route B:
Proposed Course of TcPAM from (2S)-R-Phenylalanine to (3R)-β-Phenylalaninea and Proposed Rotamers of 20-Methylcinnamate
in the TcPAM Reactiona
a The p-orbitals on the CdC and the ring are provided for perspective only and not intended to demonstrate molecular orbital concepts.
TcPAM is proposed to access a second rotamer (18A)10 from
16A to make β-phenylalanine, involving rotations about the
C1ꢀCR and CipsoꢀCβ bonds. The 20-methyl substituent of 15B
likely affected the rotation of 16B to 18B, and thus only 16B was
made by TcPAM. In contrast, PaPAM presumably utilized a
single 20-methylcinnamate rotamer (16B) to make 20-methyl-
(3S)-β-phenylalanine without encountering the torsional barrier.
The difference in product distribution between the two enzymes
supports a model consistent with steric and torsional strain
between the 20-methyl substituent and the CR-hydrogen of the
intermediary 20-methyl-(E)-cinnamate in the TcPAM reaction
(cf. rotamer 18B in Scheme 3). Notably, alternative or additive
steric interactions between the 20-methyl substrate and active-site
residues can also prevent interchange between 16B and 18B and
abort the TcPAM reaction (Scheme 3). Conversely, it can be
imagined that the single rotamer 16B on the PaPAM pathway
need not encounter the same active-site interactions to proceed
from 20-methyl-(2S)-R- (15B) to 20-methyl-(3S)-β-phenylala-
nine (6B).
’ AUTHOR INFORMATION
Corresponding Author
walke284@msu.edu
’ ACKNOWLEDGMENT
The authors thank Christopher Walsh (Harvard Medical
School) for providing the AdmH clone. This work was supported
by the National Science Foundation (CAREER Award 0746432,
K.D.W.).
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amino acids, the presumed H-bonding residues, and van der
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’ ASSOCIATED CONTENT
S
Supporting Information. Materials, gas chromatography
b
and mass spectrometry data, NMR data, experimental methods,
and instrumentation. This material is available free of charge via
the Internet at http://pubs.acs.org.
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