Mutation of Aryl Binding Pocket Residues Results in an Unexpected

Subscriber access provided by University of Cincinnati Libraries

Article

Mutation of Aryl Binding-Pocket Residues Results in an Unexpected Activity Switch in an Oryza sativa Tyrosine Aminomutase Tyler Walter, Devinda Wijewardena, and Kevin D Walker Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00331 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 3, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Mutation of Aryl Binding-Pocket Residues Results in an Unexpected Activity Switch in an Oryza sativa Tyrosine Aminomutase

Tyler Walter†, Devinda Wijewardena†, and Kevin D. Walker†‡* †

Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.

‡

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing,

MI 48824, USA. *Corresponding author email: [email protected]

Address: Michigan State University Department of Chemistry 587 S. Shaw Lane, Room 208 East Lansing, MI 48842

Telephone: 1-517-355-9715 x 257

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A 3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (TAM) isolated from the rice plant Oryza sativa (OsTAM) makes β-tyrosine (75%) and p-coumarate (25%) from α-tyrosine. OsTAM is the first TAM to have very slight native phenylalanine aminomutase (PAM) activity (3% relative to TAM activity). The active sites of OsTAM and a TcPAM from Taxus plants differ by only two residues (Y125 and N446 of OsTAM; C107 and K427 of TcPAM) positioned similarly near the aryl ring of their substrates. The kinetic parameters and substrate selectivity were measured for OsTAM single mutants Y125C- and N446K-OsTAM and double mutant Y125C/N446K-OsTAM. Compared with OsTAM, each single mutant was slower at converting αtyrosine to its β-isomer and p-coumarate; the double mutant did not produce any detectable product. Each mutant bound α-phenylalanine ~9-fold better than did OsTAM, suggesting that the mutations ஒି୔୦ୣ ୡ୧୬୬ made the catalysts more selective for phenylalanine. The total turnover rate (݇ୡୟ୲ + ݇ୡୟ୲ ) of each

mutant for converting α-phenylalanine to both β-phenylalanine and cinnamate was nearly an order of magnitude greater than the OsTAM rate for making β-phenylalanine; the latter did not make cinnamate above the limits of detection. This switch in catalytic activity from an MIO-tyrosine aminomutase (TAM) to a phenylalanine ammonia lyase (PAL) by changing only two active site side chains suggests that these residues play a central role in substrate selectivity and, in part, set the intrinsic reactivity of OsTAM.

2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Members of a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent class 1 lyase-like family are found in organisms from four of the six kingdoms of life.1-4 The MIO is formed posttranslationally and autocatalytically by condensation of an amino acid triad, (Ala/Thr/Ser/Cys)-Ser-Gly (Figure 1A), and functions as an α/β-unsaturated electrophile that facilitates α,β-elimination of the NH2/H-pair from β-aryl-α-amino acids. Members of this family include aminomutases that isomerize phenylalanine and tyrosine (PAM2 and TAM,1,5 respectively) to their corresponding β-amino acids, and ammonia lyases that eliminate NH3 from phenylalanine (PALs),6 tyrosine (TALs),7 and histidine (HALs)8 substrates to produce the corresponding trans-alkene (Figure 1B). Their chemical products are involved in diverse biochemical roles such as histidine catabolism,9 the biosynthesis of plant lignin,10 and the biosynthesis of a variety of secondary metabolites in both plants and bacteria.11,12 A

B

Figure 1. A) Condensation of tandem Ala/Thr/Ser/Cys–Ser–Gly residues to form the MIO. B) Mechanism of MIO-dependent ammonia-lyases (AL) and aminomutases (AM). Ar = p-hydroxyphenyl or phenyl.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MIO-dependent Aminomutases. MIO-dependent aminomutase begin their reactions by adding the amino group of the substrate to the methylidene carbon of the MIO prosthesis. The enzyme removes the NH2/H pair from the substrate, yielding an NH2-MIO adduct, a tightly bound acrylate intermediate, and protonated catalytic Tyr.13 The PAM and TAM enzymes typically release only ~5-10% cinnamate or p-coumarate, respectively, having lost most of their ancestral lyase activity.1,14-17 They have evolved to transfer the amino group from Cα to Cβ to form β-amino acids and reform the catalytic MIO. Other characterized MIO-aminomutases include tyrosine aminomutases from Myxococcus fulvus (MfTAM),5 Myxococcus sp. Mx-B0 (MxTAM),5 Actinomadura madurae (AmTAM),11 and Streptoalloteichus sp. ATCC 53650 (KedY4).18 Further, an MIO-dependent tyrosine aminomutase19 from Oryza sativa Japanese rice (OsTAM) isomerizes (2S)-α- to (3R)-β-tyrosine with 94% e.e. (Figure 2), while also producing an abnormally higher amount (25%) of p-coumarate as byproduct20 than made by other MIOreliant TAM enzymes.1,14 It is interesting to note that the CoA thioester of p-coumarate is known to be bacteriostatic21 and thus may provide this function in rice in addition to the role it plays in lignin and flavonoid biosynthesis.22 The high β-tyrosine enantioselectivity catalyzed by OsTAM resembles that of a Taxus canadensis phenylalanine aminomutase (TcPAM) from plants, which makes (3R)-β-phenylalanine with exceptionally high enantioselectivity (~100% e.e.).16

Figure 2. OsTAM catalyzed migration of NH2/H pair by retention-of-configuration (ROC). Tyrosine, X = OH; Phenylalanine, X = H. Substrate Selectivity and Enantioselectivity in MIO-Enzymes. Three active site residues of MIO-dependent enzymes near the para-carbon of the phenylpropanoid substrate have been 4 ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

examined in previous studies for their effect on substrate selectivity. For example, one study looked to change SgTAM (Figure 3A) to a functionally-similar phenylalanine aminomutase by changing His93 to Phe, guided by a Phe residue positioned similarly in a distantly-related plant PAL (Figure 3C and Figure 4A).23 Surprisingly, the SgTAM His93Phe mutant was not an active aminomutase with either α-phenylalanine or α-tyrosine. Formation of an acrylate byproduct (cinnamate or coumarate) was not described in the SgTAM mutant study.23 Following a similar mutation strategy in an earlier study, RsTAL from Rhodobacter sphaeroides was successfully converted to a functionally-equivalent PAL after making a His89Phe mutation.24,25 Following this recurring theme, another study changed the specificity of a TcPAM from phenylalanine to tyrosine (TcTAM) by a straightforward mutation of Cys107 (Figure 3B) to His or Ser, found in positionally–similar locations of other characterized TAMs (Figure 3A and Figure 4D).26 Overall, several studies on MIO-based aminomutases23,26 and ammonia lyases5,24,25,27 have shown that the residue in-line with the para-carbon of the phenylpropanoid substrate (e.g., Cys107 in TcPAM) (Figure 3B and Figure 4C) generally involve a histidine residue. This residue is seemingly key for controlling substrate selectivity and less for defining the intrinsic aminomutase or lyase activity.24-26


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

C

D

Figure 3. Comparison of active sites of (A) SgTAM (PDB 2RJR), (B) TcPAM (PDB 3NZ4) (each structure co-crystallized with phenylpropanoid adduct or complex (magenta)), (C) PcPAL (PDB 1W27) in complex with a cinnamate model from the TcPAM complex, and (D) OsTAM (modeled on the TcPAM structure in complex with a 4'-hydroxycinnamate model). Residues in the aryl ring binding-pocket (orange) and catalytic residues (green) are highlighted. The PcPAL structure is in an open form and its catalytic tyrosine is thus out of view. Heteroatoms are colored red for oxygen, blue for nitrogen, yellow for sulfur, and orange for fluorine of the 4'fluorophenylpropanoid in the SgTAM. Numbers listed are distances in units of Å.

Comparing the crystal structure of TcPAM with a model of OsTAM shows that their active sites differ by only two residues (Tyr125-Asn446 in OsTAM and Cys107-Lys427 in TcPAM) near the para-carbon of bound substrate (Figure 3B and 3D). Conserved residues Leu126 (OsTAM) and Leu108 (TcPAM) are in the aryl-ring binding triad (Figure 3B and 3D). Thus, we hypothesized that OsTAM could be converted to a highly enantioselective, functionally-similar OsPAM by mutating its active site to match that of TcPAM. Herein, we describe an unexpected change in function from a tyrosine aminomutase to a phenylalanine ammonia lyase. We also


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

highlight the substrate preference, kinetic parameters, and product distribution of a double (Y125C/N446K) and two single (Y125C and N446K) mutants of OsTAM.

Methods Chemicals. (3R)-β-Tyrosine and (3R)-β-phenylalanine were obtained from Peptech (Burlington, MA). All other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification, unless noted otherwise. OsTAM Mutations. Point mutations of the ostam gene were generated by the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene) using the buffers, enzymes, and dNTP mix provided. To make each single ostam mutant, the following forward and reverse primer pairs were used separately (mutated bases are underlined): Y125C: 5'-GAA CTG ATC CGT TGT CTG AAC GCT GGC-3' and 5'-GCC AGC GTT CAG ACA ACG GAT CAG TTC-3'; N446K: 5'-GAC TAT GGC TTT AAG GGT GCG GAA GTG-3' and 5'-CAC TTC CGC ACC CTT AAA GCC ATA GTC-3'. The pOsTAM plasmid containing the wild-type ostam cDNA was used as the template for PCR-amplification using Phusion HF polymerase (New England Biolabs, Ipswich, MA) under the following protocol: 95 °C for 5 min, followed by 18 cycles of 95 °C for 30 s, 58 °C for 1 min, 68 °C for 8.5 min, with a final elongation step of 68 °C for 7 min. Each PCR single mutant product plasmid (pY125C-ostam and pN446K-ostam) was digested with DpnI (New England Biolabs) at 37 °C for 1 h. The resultant pN446K-ostam plasmid was used as the template in another round of PCR with the Y125C primer set to provide the pY125C/N446Kostam double mutant plasmid. Each mutant plasmid was then used to transform DH5α Escherichia coli (Invitrogen, Thermo Life Sciences, Grand Island, NY). The resulting colonies were inoculated in starter cultures and grown for DNA purification (Wizard® Plus SV Minipreps 7 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DNA Purification System, Promega, Fitchburg, WI), and DNA sequencing to verify the ostam point mutations at the Michigan State University Research Technology Support Facility. Subcloning, Expression, and Purification of Mutant OsTAM. Mutant plasmids were used to transform Escherichia coli BL21 (DE3) cells with the pY125C-ostam, pN446K-ostam, and pY125C/N446K-ostam double mutant plasmids expressing the Y125C-OsTAM, N446KOsTAM, and Y125C/N446K-OsTAM mutant enzymes, respectively. Cells were grown in 3 L Luria-Bertani (LB) medium supplemented with kanamycin (50 µg/mL). Overexpression of the OsTAM cognates was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) (125 µM), and the cells were grown at 18 °C for 18 h. The cells were harvested by centrifugation, and the resulting pellet was resuspended in 50 mL of lysis buffer (50 mM sodium phosphate containing 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, pH 8.0). The cells were lysed by sonication (Misonix Sonicator, Farmingdale, NY), and the cellular debris was removed by centrifugation. The crude lysate was added to a nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography column (Qiagen, Valencia, CA) pre-equilibrated with wash buffer (50 mM sodium phosphate containing 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, pH 8.0), and each mutant OsTAM expressed with a His6-epitope was purified according to the protocol described by the manufacturer. Y125C-, N446K-, and Y125C/N446K-OsTAM (76 kDa) eluted separately in 250 mM imidazole fractions, were concentrated, and buffer exchanged against 50 mM sodium phosphate containing 5% (v/v) glycerol (pH 8.0) using a Centriprep centrifugal filter (30k MWCO, Millipore). The purity of the OsTAM mutants (60%, 90%, and 70%, respectively) was judged by SDS-PAGE with Coomassie Blue staining, using a Kodak Gel Logic 100 Imaging System (Figure S1-S3 of Supporting Information). Each mutant enzyme was estimated at 2.1


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mg/mL (corrected for purity) on a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE). Activity of OsTAM Mutants Tested with α-Tyrosine and α-Phenylalanine. Y125C-OsTAM, N446K-OsTAM, and Y125C/N446K-OsTAM (0.7 mg of each) were separately incubated 18 h with α-tyrosine or α-phenylalanine (2 mM) in assay buffer (50 mM sodium phosphate, 5% (v/v) glycerol, pH 8.0). The product mixture containing amino acids and acrylates was treated with pyridine (2 × 0.6 mmol) and ethyl chloroformate (2 × 0.5 mmol) in one pot and stirred for 5 min each time. For assays incubated with α-tyrosine, the resulting 4'-O,2-N- and 4'-O,3-Ndi(ethoxycarbonyl) derivatives of the α- and β-tyrosine, respectively, and any ethyl 4'-Oethoxycarbonyl derivatives of p-coumarate were extracted into diethyl ether (3 × 1 mL). The organic layer was separated and removed under a stream of nitrogen. The resultant residue was resuspended in 3:1 ethyl acetate/ethanol. For assays incubated with α-phenylalanine, the resulting 2-N- and 3-N-ethoxycarbonyl derivatives of the α- and β-phenylalanine, respectively, and cinnamate were extracted into diethyl ether (3 × 1 mL); the organic layer was separated and removed under a stream of nitrogen. The residue was resuspended in 3:1 ethyl acetate/methanol and titrated with diazomethane until a yellow color persisted. The derivatives were analyzed by gas chromatography/mass spectrometry (GC/EI-MS). The derivatized biosynthetic products had retention times and fragment ion abundances (Figure S8-S20 of the Supporting Information) identical to those of authentic standards derivatized equivalently. Kinetic Parameters of OsTAM Mutants with α-Tyrosine. Y125C- and N446K-OsTAM (0.36 mg) were incubated separately in 3 mL of assay buffer containing (2S)-α-tyrosine to establish linearity with respect to time at a fixed protein concentration at 29 °C. Note, the Y125C/N446KOsTAM double mutant was not active with α-tyrosine, so no kinetics data was acquired. 9 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

Aliquots (250 µL) were withdrawn from each at 1-h intervals over 12 h. The products were derivatized and quantified as described above, and steady state conditions were determined. To calculate the kinetic constants, (2S)-α-tyrosine was varied (0.1 – 1.5 mM) in separate assays under steady state conditions. The resultant aryl alanine and aryl acrylate products were derivatized and quantified as described earlier. Kinetic parameters (KM and kcat) were determined from Michaelis-Menten plots (Figure S21-S22 of the Supporting Information). OriginPro 9.0 was used to perform the nonlinear curve fitting. Although KM is not a true dissociation constant, in this study it will serve as a means of comparing enzyme interactions with different substrates. Kinetic Parameters of OsTAM Mutants with α-Phenylalanine. Y125C-, N446K-, and Y125C/N446K-OsTAM (0.18 mg) were incubated separately in 3 mL of assay buffer containing (2S)-α-phenylalanine to establish linearity with respect to time at a fixed protein concentration at 29 °C. Aliquots (250 µL) were withdrawn from each at 1-h intervals over 12 h. The products were derivatized and quantified as described above, and steady state conditions were determined. To calculate the kinetic constants, (2S)-α-phenylalanine was varied (0.1 – 3 mM) in separate assays under steady state conditions. The resultant aryl alanine and aryl acrylate products were derivatized and quantified as described earlier. Kinetic parameters (KM and kcat) were determined from Michaelis-Menten plots (Figure S23-S25 of the Supporting Information). OriginPro 9.0 was used to perform the nonlinear curve fitting.

Results and Discussion Activity of OsTAM Mutants. On the way to making the Y125C/N446K-OsTAM double mutant, the progenitor single mutants Y125C- and N446K-OsTAM were cloned, expressed, and tested for activity with α-tyrosine and α-phenylalanine. 10 ACS Paragon Plus Environment

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Y125C-OsTAM Mutant. Compared to OsTAM, Y125C-OsTAM turned over α-tyrosine to pcoumarate (Table 1F) an order of magnitude slower (Table 1E). While the KM of both Y125Cஒି୘୷୰ ୲୭୲ୟ୪ ୡ୧୬୬ OsTAM and OsTAM for α-tyrosine were similar (Table 1A and 1B), the ݇ୡୟ୲ (݇ୡୟ୲ + ݇ୡୟ୲ )

was drastically reduced (29-fold) for the mutant. Besides the decreased turnover, the product ratios were inverted; Y125C-OsTAM produced a 22:78 β-tyrosine to coumarate ratio compared to a reciprocal 75:25 ratio catalyzed by OsTAM. These data suggest that loss of the 4'-hydroxyl group of the Tyr125 single mutant negatively affects substrate turnover and the ability of OsTAM to retain the coumarate intermediate for the NH2-rebound on the reaction pathway. While Y125C-OsTAM produced β-phenylalanine ~5-fold slower (Table 1F) than the rate of OsTAM (Table 1E), it unpredictably gained the ability to make cinnamate (Table 1F); OsTAM did not produce detectable quantities of cinnamate (Table 1E). Also, the KM of the mutant decreased significantly for α-phenylalanine, estimating that the tandem Cys125 replacement and the original N446 of Y125C-OsTAM binds phenylalanine about 9-fold better than the Y125/N446 diad of OsTAM. The catalytic efficiency of Y125C-OsTAM for α-phenylalanine (Table 1F) was ~3-fold higher than that for α-tyrosine (Table 1B) with disproportionate partitioning to the acrylate products: 97% cinnamate from α-phenylalanine; 78% coumarate from α-tyrosine. These data support that the YC mutation changed the substrate selectivity preference to α-phenylalanine yet also affected the ability of the catalyst to hold the acrylate intermediate long enough for amine group rebound. It is interesting that while the Y125C mutation of OsTAM changes the selectivity toward phenylalanine, it disproportionates the product toward cinnamate. As mentioned, this observation is consistent with a mechanism that rapidly releases (i.e., poorly retains) the cinnamate intermediate. This is intriguing since OsTAM, with its naturally occurring diad Y125 and N446, 11 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

is less selective for phenylalanine based on its 60-fold lower relative catalytic efficiency for phenylalanine than Y125C-OsTAM (Table 1E and F). OsTAM has a 9-fold higher KM and 29୲୭୲ୟ୪ fold slower ݇ୡୟ୲ for phenylalanine compared to Y125C-OsTAM. Nonetheless, OsTAM

interestingly makes mostly β-phenylalanine and no detectable cinnamate. This suggests that Y125 and N446 work in tandem to retain the intermediate long enough for mutase catalysis, contrary to what we see from the product distribution of Y125C-OsTAM. N446K-OsTAM Mutant. The relative catalytic rate of N446K-OsTAM (Table 1C) for αtyrosine was 2.6-fold slower than the rate of Y125C-OsTAM (Table 1B) and astoundingly 76୲୭୲ୟ୪ /Krel) of N446Kfold slower than OsTAM (Table 1A). The relative catalytic efficiency (݇୰ୣ୪

OsTAM dropped a mere ~3-fold compared to that of Y125C-OsTAM and by nearly 100-fold compared to that of wild-type enzyme (Table 1). The KM values for N446K-OsTAM, Y125COsTAM, and OsTAM (Table 1A – C) are similar for tyrosine, yet it is clear that the N446K mutation clearly affects the relative proportion of tyrosine that can bind in a catalytically competent conformation. The reduce amount of product made by N446K-OsTAM from αtyrosine is comprised of a 2:98 ratio of β-tyrosine to p-coumarate (Table 1C). The predominant production of p-coumarate again supports the idea that the N466 residue is responsible, in part, for sufficiently holding the acrylate intermediate within the active site for the isomerization to advance. To compare, N446K-OsTAM turned over α-phenylalanine at a 6:94 ratio of β-phenylalanine ୲୭୲ୟ୪ to cinnamate (Table 1G) at a rate far superior (16-fold greater ݇ୡୟ୲ ) than it turned over tyrosine

to its products (Table 1C). Thus, the altered NK446 coupled with the natural Y125 residue not ୲୭୲ୟ୪ only enhanced specificity for phenylalanine (݇୰ୣ୪ /Krel is ~45-fold) over that for α-tyrosine, it

too affected the NH2-rebound, producing more of the acrylate intermediate than the β-amino 12 ACS Paragon Plus Environment

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

acid, as seen for the Y125C mutant. Moreover, the 95-fold increase and the ~100-fold decrease in catalytic efficiency of N446K-OsTAM for phenylalanine and tyrosine, respectively, compared to that of OsTAM, shows that the mutant is more compatible with a phenylpropanoid than a hydroxyphenylpropanoid. This selectivity preference was also seen for the Y125C mutant and for the double mutant, described next. Y125C/N446K-OsTAM Mutant. The double mutant Y125C/N446K-OsTAM lost all of its detectable α-tyrosine activity and did not convert the 4'-hydroxyphenylalanine to either βtyrosine or p-coumarate (Table 1D and 1H), even after 48 h. This exclusive phenylalanine selectivity, over tyrosine, is much like that of TcPAM, whose active site sequence guided the design Y125C/N446K-OsTAM. Y125C/N446K-OsTAM converted α-phenylalanine to ~40-fold more cinnamate over β-phenylalanine (Table 1H), with a total turnover rate similar to those of the single mutant congeners (Table 1F and G). Using KM as an estimate of Kd, each OsTAMmutant bound α-phenylalanine about as well as OsTAM bound α-tyrosine (Table 1A, F – H). Thus, it appears that even though Y125C/N446K-OsTAM binds α-phenylalanine as sufficiently through its three binding contacts (the aryl ring binding pocket, the amino acid NH2-MIO adduct, and the salt bridge with R344) (Figure 4) like other MIO aminomutases23,28 (e.g., see Figure 3A), the cinnamate intermediate is not well-retained by the active site after elimination of the NH2/H pair (see Figure 1B). In summary, switching Y125 and N446 of the tyrosine-specific OsTAM to C125 and K446 (residue identity found in the TcPAM) changed OsTAM to a phenylalanine-specific catalyst. In addition, these mutations changed the aminomutase reactivity to that of an ammonia lyase. Thus, the Y125 and N446 residues of OsTAM are key to binding the tyrosine substrate and in part defining the reaction type (aminomutase or lyase). These residues near the 4'-hydroxy of the 13 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 24

substrate likely interact through polar interactions and hydrogen bonding to control the capture time of the acrylate intermediate. Moreover, characterized bacterial TAMs hypothetically use 3 to 4 polar active site residues: His-Ser-[Glu/Ala]-Tyr (underlined) to aid in binding their 4'hydroxyphenylalanine substrate (Figure 3A and Figure 4D). By contrast, OsTAM uses only two: Tyr125-Leu126-Asn446-Val450 (underlined), respectively (Figure 3D and Figure 4A). The reduced number of polar contacts with the tyrosine substrate may provide insight into why the proportion of p-coumarate-release increased and why the KM of OsTAM was higher for tyrosine compared to that of previously characterized TAMs. Table 1. Kinetic Parameters of Wild-type and Mutant OsTAM Enzymes. OsTAM Enzyme Wild-type

A

Y125C

B

N446K

C

Y125C/N446K D

OsTAM Enzyme

KM (mM) 0.54 (0.03) 0.51 (0.06) 0.64 (0.08)

Substrate: α-Tyrosine a ஒି୘୷୰ େ୭୳୫ -1 b ୲୭୲ୟ୪ -1 d (s ) ݇ୡୟ୲ (s-1) ݇ୡୟ୲ c ݇ୡୟ୲ (s ) β-Tyr:Coum 3 3 × 10 × 103 × 10 5.0 1.7 75:25 6.7 (0.014) (0.003) 0.053 0.18 22:78 0.23 (0.003) (0.007) 0.0021 0.086 2:98 0.088 (0.0002) (0.007)

g

ND

KM (mM)

ND

E

Y125C

F

N446K

G

Y125C/N446K H

---

Substrate: α-Phenylalanine େ୧୬୬ -1 j ୲୭୲ୟ୪ -1 d (s ) ݇ୡୟ୲ (s ) c ݇ୡୟ୲ (s ) β-Phe:Cinn 3 × 10 × 103 × 103 h ND *0.16 100:0 0.16 (0.046) 1.2 0.035 3:97 1.24 (0.17) (0.003) 1.3 0.08 6:94 1.38 (0.05) (0.009) 2.0 0.052 2:98 2.05 (0.24) (0.005)

ஒି୔୦ୣ ݇ୡୟ୲

h

Wild-type

ND

*9.0 (2.4) 1.0 (0.21) 0.72 (0.10) 0.49 (0.11)

-1 i

୲୭୲ୟ୪ ݇୰ୣ୪ e (%)

୲୭୲ୟ୪ ୲୭୲ୟ୪ /Krel ݇ୡୟ୲ /KM ݇୰ୣ୪ -1 -1 f (s M ) (%)

100

12.4

100

3.4

0.45

3.6

1.3

0.133

1.1

---

---

---

୲୭୲ୟ୪ ݇୰ୣ୪ e (%)

୲୭୲ୟ୪ ୲୭୲ୟ୪ ݇ୡୟ୲ /KM ݇୰ୣ୪ /Krel f (s-1 M-1) (%)

7.8

0.02

0.48

60

1.24

29

67

1.91

45

100

4.21

100

kcat of enzyme for β-tyrosine. kcat of enzyme for coumarate. Ratio of β-amino acid to acrylate product. Total turnover of enzyme for β-amino acid and acrylate products; eRelative total turnover. fRatio of relative catalytic efficiency. gND = not detected; limit of detection estimated at 0.000075 × 10-3 s-1. hNonlinear regression of the turnover rate of OsTAM for α-phenylalanine did not reach saturation. Thus, apparent Km and kcat were estimated from a linear regression (Lineweaver-Burk) plot.20 ikcat of enzyme for β-tyrosine. jkcat of enzyme for coumarate. Standard error given in parentheses a

b

c

d


Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

98 | 122 | 188 | 338 | 444 A OsTAM:..YGVTTGF..|..LIRYLNAG..|..GTITASGDLVPL..|..QDRYAIR..|..GFNG CpPAL:..YGVTTGF..|..LIRFLNAG..|..GTITASGDLVPL..|..QDRYALR..|..GFKG PcPAL:..YGVTTGF..|..LIRFLNAG..|..GTITASGDLVPL..|..QDRYALR..|..GFKG AtPAL:..YGVTTGF..|..LIRFLNAG..|..GTITASGDLVPL..|..QDRYALR..|..GFKG OsPAL:..YGVTTGF..|..LIRFLNAG..|..GTITASGDLVPL..|..QDRYALR..|..GFKG ZmPAL:..YGVTTGF..|..LLRHLNAG..|..GTITASGDLVPL..|..QDRYALR..|..GFKG B RsTAL:..YGLTTGF..|..LVHHLASG..|..GTVGASGDLTPL..|..QDAYSLR..|..GFMG RcTAL:..YGITTGF..|..LIYHLATG..|..GTVGASGDLTPL..|..QDAYSLR..|..GFMG SeTAL:..YGLTQGF..|..LISHLGTG..|..GTVSASGDLQPL..|..QEPYSLR..|..GLAG C TcPAM:..YGVTTGF..|..LIRCLLAG..|..GSVSASGDLIPL..|..QDRYALR..|..GLKG PaPAM:..YGVNTSM..|..LINAVATN..|..GSLGTSGDLGPL..|..EDAYSIR..|..GLMG EncP :..YGVNTSM..|..LINAVATN..|..GSLGTSGDLGPL..|..EDAYSIR..|..GLMG D SgTAM:..YGVTTGY..|..LVRSHSAG..|..GSLGASGDLAPL..|..QKAYSLR..|..GFAG AmTAM:..YGVTTGY..|..LVRSHSAG..|..GSLGASGDLAPL..|..QKAYSLR..|..GFAG MfTAM:..YGVNTGF..|..LLRSHAAG..|..GSLGASGDLAPL..|..QNAYSLR..|..GFEG CcTAM:..YGVNTGF..|..LIRSHAAG..|..GSLGASGDLSPL..|..QDAYTLR..|..GFEG

Figure 4. Partial sequence alignment (GeneDoc 2.6, BLOSUM 62) of MIO-dependent enzymes. Conserved residues (green) are catalytic Tyr, the Ala/Thr-Ser-Gly triad (which form the MIO group), and Arg (forms salt-bridge with the carboxylate of the substrate). Residues proposed as being key for substrate selectivity (highlighted in yellow with bold text). Residue numbering based on OsTAM. Aminomutases (AMs) and ammonia lyases (ALs) from the following genus species are listed: (A) CpPAL, Cenchrus purpureus (Accession No. KF447872); PcPAL, Petroselinum crispum (Accession No. P24481); AtPAL, Arabidopsis thaliana (Accession No. AAP59438); OsPAL, Oryza sativa (Accession No. CAA61198); ZmPAL, Zea mays (Accession No. AAL40137); (B) RsTAL, Rhodobacter sphaeroides (Accession No. Q3IWB0); RcTAL, Rhodobacter capsulatus (Accession No. WP_023923512); SeTAL, Saccharomyces expedia (Accession No. ABC88669); (C) TcPAM, Taxus canadensis (Accession No. AY582743); PaPAM, Pantoea agglomerans (Accession No. AAO39102); EncP, Streptomyces maritimus (Accession No. AAF81735); (D) SgTAM, Streptomyces globisporus (Accession No. Q8GMG0); AmTAM, Actinomadura madurae (Accession No. ABY66005); MfTAM, Myxococcus fulvus (Accession No. B8ZV93); CcTAM, Chrondromyes crocatus (Accession No. Q0VZ68). Single letter designations for phenylalanine (P) and tyrosine (T) are used in enzyme abbreviations. Sequence Analysis of OsTAM with Other MIO-Dependent Enzymes. Comparison with Ammonia Lyase and Aminomutases. Aligning OsTAM with plant PALs (CpPAL, PcPAL, AtPAL, OsPAL, ZmPAL) (Figure 4A) shows high sequence identity between 63% (for AtPAL from Arabidopsis thaliana) and as high as 73% (for CpPAL from Cenchrus purpureus); the sequence similarity ranged between 77% and 81%. It was surprising to find that OsTAM had low sequence identity (19 – 20%) and similarity (29 – 31%) with other TAMs


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

(Figure 4D). These sequence comparisons infer that OsTAM likely arises from a close ancestral plant MIO-AL rather than from a plant MIO-AM. An earlier study found that OsTAM had no detectable PAL activity, but did release an unusually elevated proportion of p-coumarate byproduct (25%), through its TAL activity.20 Other MIO-aminomutases release less acrylate,2 for example, TcPAM releases 5-10% byproduct (thereby less AL activity) at 31 °C during its reaction at steady state.1,2,14 This reduced AL activity for TcPAM could correlate with its lower sequence overlap (41-43% identity, 59-62% similarity) (Figure 4C) when compared with PAL sequences. Mutational Analyses. Previous investigations looked to switch the activities of MIOdependent aminomutases and ammonia-lyases.5,29-31 One study showed how increasing the reaction temperature promoted ammonia-lyase activity catalyzed by PaPAM isolated from Pantoea agglomerans.31 A separate study switched a PAM to a PAL by mutating four residues (Leu97, Ala77, Ile79, and Cys89) of the inner loop structure of Taxus chinensis (TchPAM) [essentially identical (97%) in sequence to TcPAM from Taxus canadensis] to those in PcPAL (Gly127, Thr107, Ser109, and Thr119, respectively).30 The sequence of the same loop region of OsTAM is 54% identical to the one in TcPAM and 66% identical to the one in PcPAL. This suggests further that OsTAM bears more intrinsic ammonia lyase character. Further, the four key inner loop residues in OsTAM (Phe115, Thr95, Thr97, and Ala107, respectively, described above) show greater similarity to the four positionally-related residues in PcPAL than in TcPAM. Early mutational studies on the inner loop of TcPAM suggest that the hydrophobic residue (Ile79) maintains a functional inner loop framework.30 An Ile79Ser-TcPAM mutant made a larger proportion of cinnamate over β-phenylalanine compared to that made by TcPAM. 16 ACS Paragon Plus Environment

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

OsTAM, by comparison, has a hydrophilic Thr97 residue and is imagined to lower the ability of the inner loop to retain the acrylate intermediate, thus preventing it from retaining p-coumarate as well as other MIO-TAMs.1,14 We note that a likely paradox exists if the hydrophilic Thr97 residue of the inner loop of the wild-type OsTAM is presumed to reduce the retention of the acrylate intermediate on the aminomutase pathway. Phenylalanine is an inherently weakerbinding substrate for OsTAM (Table 1E) when the residues near the phenyl ring binding pocket are Y125 and N446. Thus, if the loop structure of OsTAM is ammonia lyase-like and preferentially releases the acrylate intermediate along the reaction pathway, we would expect OsTAM to selectively release cinnamate intermediate before the NH2-rebound occurs to form βphenylalanine. Thus, the combined effects of the loop structure and the residues that bind the aryl ring in the active sites on the mechanism of MIO-dependent aminomutases remain obscure.

Conclusion This study looked to further understand the determinants of MIO-dependent catalysts that define substrate selectivity, binding affinity, turnover rate, and ammonia lyase or aminomutase activity. The objective was to determine the effect on substrate selectivity after mutations of a triad (Tyr125-Leu126-Asn446) in the aryl ring binding-pocket in OsTAM. This triad is unique from the consensus sequence (Ser-His-Glu/Ala) of several other MIO-TAMs (Figure 4D) and is more similar to the one (Phe/His-Leu-X) found in MIO-dependent ALs (Figure 4A and 4B). Mutation of Asn446 of OsTAM to a Lys residue created an active site bearing the characteristic triad sequence ([Aromatic Amino Acid]-Leu-Lys) conserved in all PALs, and thus could explain in part why N446K-OsTAM had increased PAL activity over that of OsTAM. Likewise, the Y125C/N446K-OsTAM mutant was designed so its active site exactly matched the TcPAM


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

active site. We imagined that Y125C/N446K-OsTAM would function chiefly as an OsPAM, but instead, we found that it principally catalyzed a PAL reaction at a rate similar to that of N446KOsTAM, showing only 2% PAM reactivity. This investigation demonstrates a unique instance of mutating two binding pocket residues of a TAM to convert it into a PAL, and thus provides critical insight into the role these key residues may play with the inner loop structure that caps the active site. These residues in combination with the loop structure likely increase the residence time of the acrylate intermediate in the active site for the NH2-rebound step.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:XXXXXXXX Instrumentation, experimental methods, SDS-PAGE protein documents, and GC/MS and kinetics data (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Funding The authors are grateful to the MSU Office for Inclusion and Intercultural Initiatives for supporting the MSU Chemistry 4-Plus Bridge to the Doctorate Program (GA017081-CIEG) Notes The authors declare no competing financial interest.


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ACKNOWLEGMENTS We thank Gayanthi Attanayake for her technical assistance. Graphical Abstract X = OH

X=H

OsTAM

Y125C/N446K-OsTAM 73% krel = 5.0 0% krel = 0.0

2% krel = 0.16 3% krel = 0.05

25% krel = 1.7 0% krel = 0.0

0% krel = 0.0 97% krel = 2.0


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

REFERENCES

1.

Christianson, C. V., Montavon, T. J., Van Lanen, S. G., Shen, B., and Bruner, S. D. (2007) The structure of L-tyrosine 2,3-aminomutase from the C-1027 enediyne antitumor antibiotic biosynthetic pathway, Biochemistry 46, 7205-7214.

2.

Feng, L., Wanninayake, U., Strom, S., Geiger, J., and Walker, K. D. (2011) Mechanistic, mutational, and structural evaluation of a Taxus phenylalanine aminomutase, Biochemistry 50, 2919-2930.

3.

Suchi, M., Harada, N., Wada, Y., and Takagi, Y. (1993) Molecular cloning of a cDNAencoding human histidase, Biochim. Biophys. Acta 1216, 293-295.

4.

Babich, O. O., Pokrovsky, V. S., Anisimova, N. Y., Sokolov, N. N., and Prosekov, A. Y. (2013) Recombinant L-phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent, Biotechnol. Appl. Biochem. 60, 316-322.

5.

Krug, D., and Mueller, R. (2009) Discovery of additional members of the tyrosine aminomutase enzyme family and the mutational analysis of CmdF, ChemBioChem 10, 741-750.

6.

MacDonald, M. J., and D'Cunha, G. B. (2007) A modern view of phenylalanine ammonia lyase, Biochem. Cell Biol. 85, 273-282 (and references therein).

7.

Kyndt, J. A., Meyer, T. E., Cusanovich, M. A., and Van Beeumen, J. J. (2002) Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein, FEBS Lett. 512, 240-244.


Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

8.

Baedeker, M., and Schulz, G. E. (2002) Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism, Eur. J. Biochem. 269, 17901797.

9.

Schwede, T. F., Retey, J., and Schulz, G. E. (1999) Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile, Biochemistry 38, 5355-5361.

10.

Hahlbrock, K., and Scheel, D. (1989) Physiology and molecular biology of phenylpropanoid metabolism, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 347-369.

11.

Van Lanen, S. G., Oh, T.-j., Liu, W., Wendt-Pienkowski, E., and Shen, B. (2007) Characterization of the maduropeptin biosynthetic gene cluster from Actinomadura madurae ATCC 39144 supporting a unifying paradigm for enediyne biosynthesis, J. Am. Chem. Soc. 129, 13082-13094.

12.

Walker, K. D., Klettke, K., Akiyama, T., and Croteau, R. (2004) Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis, J. Biol. Chem. 279, 53947-53954.

13.

Wanninayake, U., and Walker, K. D. (2012) Assessing the deamination rate of a covalent aminomutase adduct by burst phase analysis, Biochemistry 51, 5226-5228.

14.

Wanninayake, U., and Walker, K. D. (2013) A bacterial tyrosine aminomutase proceeds through retention or inversion of stereochemistry to catalyze its isomerization reaction, Journal of the American Chemical Society 135, 11193-11204.

15.

Mutatu, W., Klettke, K. L., Foster, C., and Walker, K. D. (2007) Unusual mechanism for an aminomutase rearrangement: Retention of configuration at the migration termini, Biochemistry 46, 9785-9794.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16.

Page 22 of 24

Klettke, K. L., Sanyal, S., Mutatu, W., and Walker, K. D. (2007) β-Styryl- and β-aryl-βalanine products of phenylalanine aminomutase catalysis, J. Am. Chem. Soc. 129, 69886989.

17.

Ratnayake, N. D., Wanninayake, U., Geiger, J. H., and Walker, K. D. (2011) Stereochemistry and mechanism of a microbial phenylalanine aminomutase, J. Am. Chem. Soc. 133, 8531-8533.

18.

Huang, S.-X., Lohman, J. R., Huang, T., and Shen, B. (2013) A new member of the 4methylideneimidazole-5-one–containing

aminomutase

family from

the

enediyne

kedarcidin biosynthetic pathway, Proc. Natl. Acad. Sci. U. S. A. 110, 8069-8074. 19.

Yan, J., Aboshi, T., Teraishi, M., Strickler, S. R., Spindel, J. E., Tung, C. W., Takata, R., Matsumoto, F., Maesaka, Y., McCouch, S. R., Okumoto, Y., Mori, N., and Jander, G. (2015) The tyrosine aminomutase TAM1 is required for β-tyrosine biosynthesis in rice, Plant Cell 27, 1265-1278.

20.

Walter, T., King, Z., and Walker, K. D. (2016) A tyrosine aminomutase from rice (Oryza sativa) isomerizes (S)-α- to (R)-β-tyrosine with unique high enantioselectivity and retention of configuration, Biochemistry 55, 1-4.

21.

Parke, D., and Ornston, L. N. (2004) Toxicity caused by hydroxycinnamoyl-coenzyme A thioester accumulation in mutants of Acinetobacter sp. strain ADP1, Appl. Environ. Microbiol. 70, 2974-2983.

22.

Sun, H., Li, Y., Feng, S., Zou, W., Guo, K., Fan, C., Si, S., and Peng, L. (2013) Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice, Biochem. Biophys. Res. Commun. 430, 1151-1156.


Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

23.

Bruner, S. D., and Cooke, H. A. (2010) Probing the active site of MIO-dependent 2,3aminomutases, key catalysts in the biosynthesis of β-amino acids incorporated in secondary metabolites, Biopolymers 93, 802-810.

24.

Watts, K. T., Mijts, B. N., Lee, P. C., Manning, A. J., and Schmidt-Dannert, C. (2006) Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family, Chem. Biol. 13, 1317-1326.

25.

Louie, G. V., Bowman, M. E., Moffitt, M. C., Baiga, T. J., Moore, B. S., and Noel, J. P. (2006) Structural determinants and modulation of substrate specificity in phenylalaninetyrosine ammonia-lyases, Chem. Biol. 13, 1327.

26.

Wu, B., Szymanski, W., Wijma, H. J., Crismaru, C. G., de Wildeman, S., Poelarends, G. J., Feringa, B. L., and Janssen, D. B. (2010) Engineering of an enantioselective tyrosine aminomutase by mutation of a single active site residue in phenylalanine aminomutase, Chem. Commun. 46, 8157-8159.

27.

Bartsch, S., and Bornscheuer, U. T. (2009) A single residue influences the reaction mechanism of ammonia lyases and mutases, Angew. Chem., Int. Ed. Engl. 48, 3362-3365.

28.

Strom, S., Wanninayake, U., Ratnayake, N. D., Walker, K. D., and Geiger, J. H. (2012) Insights into the mechanistic pathway of the Pantoea agglomerans phenylalanine aminomutase, Angew. Chem. Int. Ed. Engl. 51, 2898-2902.

29.

Bartsch, S., Wybenga, G. G., Jansen, M., Heberling, M. M., Wu, B., Dijkstra, B. W., and Janssen, D. B. (2013) Redesign of a phenylalanine aminomutase into a phenylalanine ammonia lyase, Chemcatchem 5, 1797-1802.

30.

Heberling, M. M., Masman, M. F., Bartsch, S., Wybenga, G. G., Dijkstra, B. W., Marrink, S. J., and Janssen, D. B. (2015) Ironing out their differences: Dissecting the


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

structural determinants of a phenylalanine aminomutase and ammonia lyase, Acs Chem Biol 10, 989-997. 31.

Chesters, C., Wilding, M., Goodall, M., and Micklefield, J. (2012) Thermal bifunctionality of bacterial phenylalanine aminomutase and ammonia lyase enzymes, Angew. Chem. Int. Ed. 51, 4344-4348.


Page 24 of 24

Mutation of Aryl Binding Pocket Residues Results in an Unexpected

Recommend Documents