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`csh/PROSCI/111782/ps0520594
`
`Structural basis for the enantiospecificities of R- and
`S-specific phenoxypropionate/a-ketoglutarate dioxygenases
`
`TINA A. MU¨ LLER,1 MARIA I. ZAVODSZKY,2 MICHAEL FEIG,2,3 LESLIE A. KUHN,2
`AND ROBERT P. HAUSINGER1,2
`Departments of 1Microbiology & Molecular Genetics, 2Biochemistry & Molecular Biology, and 3Chemistry, Michigan
`State University, East Lansing, Michigan 48824-4320, USA
`
`(RECEIVED December 21, 2005; FINAL REVISION March 15, 2006; ACCEPTED March 19, 2006)
`
`Abstract
`
`(R)- and (S)-dichlorprop/a-ketoglutarate dioxygenases (RdpA and SdpA) catalyze the oxidative cleavage of
`2-(2,4-dichlorophenoxy)propanoic acid (dichlorprop) and 2-(4-chloro-2-methyl-phenoxy)propanoic acid (meco-
`prop) to form pyruvate plus the corresponding phenol concurrent with the conversion of a-ketoglutarate
`(aKG) to succinate plus CO2. RdpA and SdpA are strictly enantiospecific, converting only the (R) or the (S)
`enantiomer, respectively. Homology models were generated for both enzymes on the basis of the structure
`of the related enzyme TauD (PDB code 1OS7). Docking was used to predict the orientation of the appro-
`priate mecoprop enantiomer in each protein, and the predictions were tested by characterizing the activities
`of site-directed variants of the enzymes. Mutant proteins that changed at residues predicted to interact with
`(R)- or (S)-mecoprop exhibited significantly reduced activity, often accompanied by increased Km values,
`consistent with roles for these residues in substrate binding. Four of the designed SdpA variants were
`(slightly) active with (R)-mecoprop. The results of the kinetic investigations are consistent with the iden-
`tification of key interactions in the structural models and demonstrate that enantiospecificity is coordinated
`by the interactions of a number of residues in RdpA and SdpA. Most significantly, residues Phe171 in
`RdpA and Glu69 in SdpA apparently act by hindering the binding of the wrong enantiomer more than the
`correct one, as judged by the observed decreases in Km when these side chains are replaced by Ala.
`
`Keywords: dioxygenase; enantiospecificity; mecoprop; site-directed mutagenesis; structural modeling;
`docking
`
`Supplemental material: see www.proteinscience.org
`
`Reprint requests to: Robert P. Hausinger, Department of Microbiol-
`ogy and Molecular Genetics, 6193 Biomedical Physical Sciences,
`Michigan State University, East Lansing, MI 48824-4320, USA;
`e-mail: hausinge@msu.edu; fax: (517) 353-8957.
`Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; AlkB, alkylation-
`damaged DNA repair enzyme; AtsK, alkyl sulfatase; ANS, anthocyanidin
`synthase; CarC, carbapenam synthase; CAS, clavaminate synthase; CSD,
`Cambridge Structural Database; DAOCS, deacetoxycephalosporin C syn-
`thase; dichlorprop, 2-(2,4-dichlorophenoxy)propanoic acid; FIH, factor
`inhibiting hypoxia-inducible factor; aKG, a-ketoglutarate; mecoprop,
`2-(4-chloro-2-methyl-phenoxy)propanoic acid; NTA, nitrilotriacetic acid;
`PAHX, phytanoyl-coenzyme A 2-hydroxylase; PDB, Protein Data Bank;
`(R)-specific dichlorprop/aKG dioxygenase; SdpA,
`(S)-specific
`RdpA,
`dichlorprop/aKG dioxygenase; TfdA, 2,4-D/aKG dioxygenase; TauD,
`taurine/aKG dioxygenase; TR, tropinone reductase; SDS-PAGE, sodium
`dodecyl sulfate-polyacrylamide gel electrophoresis.
`Article and publication are at http://www.proteinscience.org/cgi/doi/
`10.1110/ps.052059406.
`
`Phenoxyalkanoic acids are systemic and post-emergence
`inhibitors of broadleaf weeds and are among the most
`widely applied herbicides in the world (Worthing and
`Hance 1991; Ahrens 1994; Donaldson et al. 2002). These
`synthetic auxins (A˚ berg 1973; Loos 1975; Ahrens 1994)
`include 2,4-dichlorophenoxyacetic acid (2,4-D) along
`with the chiral representatives 2-(2,4-dichlorophenoxy)-
`propanoic acid (dichlorprop) and 2-(4-chloro-2-methyl-
`phenoxy)propanoic acid (mecoprop), of which only the
`(R)-enantiomers are herbicidally active (Matell 1953). Micro-
`organisms able to degrade these phenoxyalkanoic acid
`herbicides have been isolated from different environments,
`and their degradative pathways have been elucidated
`(Zipper et al. 1996; Hausinger et al. 1997; Tett et al. 1997;
`
`1356
`
`Protein Science (2006), 15:1356–1368. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2006 The Protein Society
`
`ps0520594 Mu¨ller et al. ARTICLE RA
`
`Inari Ex. 1020
`Inari Agric. v. Corteva Agriscience
`PGR2023-00022
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`JOBNAME: PROSCI 15#6 2006 PAGE: 2 OUTPUT: Friday May 12 01:28:15 2006
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`Mu¨ller et al. 1999, 2001). For example, the first step in 2,4-
`D metabolism is side-chain hydroxylation to form an un-
`stable intermediate that decomposes by elimination of the
`phenol derivative (Scheme 1).
`The 2,4-D hydroxylase (TfdA) from Cupriavidus necator
`(formerly Ralstonia eutropha) JMP134(pJP4) has been
`intensively studied and shown to require FeII as a cofactor
`and a-ketoglutarate (aKG) as a cosubstrate (Fukumori and
`Hausinger 1993a,b; Saari and Hausinger 1998; Hegg et al.
`1999; Hogan et al. 2000; Dunning Hotopp and Hausinger
`2002). For mecoprop or dichlorprop metabolism, best studied
`in the soil bacterium Sphingomonas herbicidovorans MH,
`the enantiomers are separately transported into the cell by
`distinct uptake systems (Nickel et al. 1997), and enantiomer-
`(R)-
`and (S)-dichlorprop/aKG dioxygenases
`specific
`(RdpA and SdpA) catalyze the initial degradation steps as
`illustrated in Scheme 2 (Nickel et al. 1997; Mu¨ller 2004;
`Mu¨ller et al. 2004b). RdpA and SdpA share 30% amino
`acid sequence identity to each other and 30% and 37%
`identity, respectively, to TfdA, with no significant gaps in
`alignment quality, indicating that they are all close struc-
`tural homologs (Sander and Schneider 1991). The substituted
`phenol products released from these FeII/aKG-dependent
`dioxygenases are subsequently converted to the corresponding
`catechols and further metabolized by the modified ortho-
`cleavage pathway.
`The herbicide-degrading dioxygenases belong to a large
`family of mononuclear, nonheme FeII enzymes that catalyze
`a broad array of reactions (for review, see Hausinger 2004;
`Clifton et al. 2006) including hydroxylations, epoxidations,
`desaturations, ring formation, ring expansion, and as only
`recently discovered, chlorinations
`(Vaillancourt et al.
`2005a,b). Crystal structures have been elucidated for several
`family members including taurine/aKG dioxygenase (TauD)
`(Elkins et al. 2002; O’Brien et al. 2003), alkyl sulfatase
`(AtsK) (Mu¨ller et al. 2004a, 2005), clavaminate synthase
`(CAS) (Zhang et al. 2000), deacetoxycephalosporin C syn-
`thase (DAOCS) (Valega˚rd et al. 1998), anthocyanidin syn-
`thase (ANS) (Wilmouth et al. 2002), carbapenam synthase
`(CarC) (Clifton et al. 2003), proline 3-hydroxylase (Clifton
`et al. 2001), the factor inhibiting hypoxia-inducible factor
`(FIH) (Dann et al. 2002; Elkins et al. 2003), phytanoyl-
`
`Dichlorprop hydroxylase enantiospecificity
`
`coenzyme A 2-hydroxylase (PAHX) (McDonough et al.
`2005), and the DNA repair enzyme AlkB (Yu et al. 2006).
`The structures reveal a common b-jelly roll or double-
`stranded b-helix fold containing a metal ion-binding motif:
`His1-X-Asp/Glu-Xn-His2 (where n varies from 40 to 153
`residues). Three water molecules occupy the remaining
`metal ligand positions in the resting enzyme. Two water
`molecules are displaced upon binding of the cosubstrate,
`with the aKG C-2 keto group coordinating opposite the
`carboxylate side chain and the aKG C-1 carboxyl group
`binding opposite either His1 (TauD, AtsK, CAS, and FIH)
`or opposite His2 (DAOCS, ANS, CarC, PAHX, and AlkB),
`with a nearby Arg residue (located 15–22 residues beyond
`His2 in the sequence) providing additional stabilization to
`the C-1 carboxylate in the cases of TauD, AtsK, CAS, CarC,
`and AlkB. Another Arg residue (located in the sequence
`about 10 residues beyond His2) is positioned to form an ion
`pair with the C-5 carboxylate of aKG in all structures
`except FIH, where a Lys located elsewhere in the sequence
`provides stabilization. Unlike other FeII sites, the aKG-
`bound metallocenters exhibit a characteristic metal-to-ligand
`charge-transfer transition (Pavel et al. 1998; Hegg et al.
`1999; Ryle et al. 1999; Trewick et al. 2002) conferring a
`lilac color to this state of the enzymes. The primary sub-
`strate (e.g., taurine in the case of TauD) does not bind to the
`metal center, but the aforementioned crystallographic stud-
`ies and additional spectroscopic evidence (Ho et al. 2001;
`Zhou et al. 2001) indicate that substrate binding leads to the
`loss of the final water molecule, thus creating a site for
`binding of oxygen. In the case of TauD, oxidative decar-
`boxylation of aKG has been shown to produce an FeIV-oxo
`intermediate species that inserts oxygen into the unacti-
`vated C–H bond (Price et al. 2003a,b, 2005; Proshlyakov et al.
`2004; Riggs-Gelasco et al. 2004; Grzyska et al. 2005).
`Sequence alignments highlight several potential key
`residues of the (R)- and (S)-dichlorprop/aKG dioxygen-
`ases from S. herbicidovorans MH (Mu¨ ller 2004). The
`His1-X-Asp/Glu-Xn-His2 motif of RdpA is comprised of
`residues His111, Asp113, and His270, while that of SdpA
`involves His102, Asp104, and His257. Fifteen residues
`beyond His2 are residues predicted to interact with the
`C-1 carboxylate of aKG, Arg285 and His272, respectively.
`
`Scheme 1.
`
`www.proteinscience.org
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`1357
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`
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`JOBNAME: PROSCI 15#6 2006 PAGE: 3 OUTPUT: Friday May 12 01:28:16 2006
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`
`Mu¨ ller et al.
`
`Scheme 2.
`
`Furthermore, Arg281 and Arg268 of the two proteins are
`predicted to form salt bridges to the aKG C-5 carboxylate,
`with additional interactions involving Thr138 of RdpA and
`Thr129 of SdpA. In contrast to these conserved residues,
`essentially nothing is known about the phenoxypropanoic
`acid-binding sites of these proteins, especially with regard
`to the structural basis of enantiospecificity.
`Here, we describe the construction of homology mod-
`els of SdpA and RdpA from S. herbicidovorans MH and
`the use of docking to identify residues likely to be
`involved in herbicide binding. Previous homology models
`have led to successes in elucidating or designing speci-
`ficity-conferring interactions in ligands. For instance,
`homology modeling of a cercarial (human parasite) elastase
`led to the development of an effective elastase inhibitor
`(Cohen et al. 1991) and to understanding the specificity
`determinants for ligands binding to a parasite tRNA synthe-
`tase versus its human homolog (Sukuru et al. 2006). Here,
`we test by site-directed mutagenesis and kinetic analysis
`the residues predicted to be involved in substrate binding,
`enantiospecificity, or catalysis. The activity experiments are
`consistent with the key residues identified by modeling
`being involved in substrate binding. We provide additional
`evidence that several amino acids are responsible for the
`enantiospecificity of RdpA and SdpA, demonstrate that the
`active site of SdpA is less specific than RdpA for its
`substrate, and discuss the structural implications of these
`results.
`
`Results
`
`SdpA and RdpA homology models
`
`RdpA and SdpA were aligned with TauD (Supplemental
`Fig. S1), and homology models were created as described
`in Materials and Methods (Supplemental Fig. S2) using
`
`1358
`
`Protein Science, vol. 15
`
`the TauD structure as a structural template (O’Brien et al.
`2003). The two phenoxypropionate-degrading proteins
`are predicted to contain jelly roll or double-stranded
`b-helix folds comprised of eight b-strands with connect-
`ing loops, as is typical of this enzyme family (Hausinger
`2004; Clifton et al. 2006). The homology models contain
`FeII-binding sites (His111, Asp113, and His270 in RdpA
`or His102, Asp104, and His257 in SdpA), as expected
`from former sequence alignments with other FeII/aKG-
`dependent dioxygenases (Mu¨ ller 2004). The high degree
`of active-site sequence identity and strong orientation of
`key side chains by interactions with the FeII are support-
`ive of the active site being the most conserved and struc-
`turally accurate part of the RdpA and SdpA models. Detailed
`analysis of favored aKG-binding motifs in other members
`of this enzyme family indicate the iron is chelated by aKG
`with its keto group positioned opposite Asp113 of RdpA or
`Asp104 of SdpA and its C-1 carboxylate located so as to
`interact with His111 and His102, respectively. The posi-
`tively charged residues Arg285 in RdpA and His272 in
`SdpA are well positioned to provide additional stabilization
`of the aKG C-1 carboxylate, and, in each protein, the C-5
`carboxylate of aKG forms a salt bridge with Arg268 and
`Arg281, respectively. Whereas the RdpA structure repre-
`sents only one subunit of the predicted trimeric protein,
`SdpA is suggested to be monomeric, based on gel-filtration
`experiments (Mu¨ller 2004).
`
`Docking of substrates into the RdpA and SdpA structures
`
`(R)- and (S)-mecoprop were
`The natural substrates
`docked into the active sites of RdpA and SdpA to gain
`insight into the basis of enzyme enantiospecificity. First,
`the cosubstrate aKG was modeled into the active sites of
`RdpA and SdpA using two distinct conformations, as found in
`the crystal structures of FeII/aKG-dependent dioxygenases
`
` 1469896x, 2006, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1110/ps.052059406, Wiley Online Library on [27/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
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`
`Dichlorprop hydroxylase enantiospecificity
`
`(Clifton et al. 2006). The flat conformation has the five-
`member ring formed by the metal chelate coplanar with the
`C-5 carboxylate, whereas the twist conformation has the
`two planes forming a 90° angle. The resulting four models
`were energy-minimized and used as targets for substrate
`docking with the program SLIDE (Zavodszky et al. 2002)
`with the assumption that the substrate carbon undergoing
`hydroxylation would be located approximately at the same
`position relative to the iron center as the key carbon atom of
`taurine in TauD (Elkins et al. 2002; O’Brien et al. 2003).
`The mecoprop-docking interactions with RdpA and SdpA
`were analyzed in detail, and one model of each protein was
`selected based on the most favorable interactions between
`enzyme and substrate (see below). These models are
`illustrated in Figure 1, with the corresponding plots of
`mecoprop interactions shown in Figure 2.
`
`Binding of (R)-mecoprop to RdpA
`
`The substrate (R)-mecoprop consists of a hydrophobic
`phenoxy ring and a polar propanoic acid, with both
`components needing to be accommodated and bound by
`the active site. The aKG conformation leading to the
`most favorable interactions has aKG in the twist confor-
`
`mation and positions the phenoxy ring of (R)-mecoprop
`as illustrated in Figure 1A (with the corresponding inter-
`actions plotted in Fig. 2A). The mecoprop carboxylate
`interacts with the amide nitrogen of Ser114, the hydroxyl
`group of Tyr221, and a guanidino nitrogen of Arg285. The
`Tyr221 hydroxyl group also is predicted to lie near (3.5 A˚ )
`the substrate ether oxygen atom and could play a role in
`directing enantiospecificity. Residues lining the hydropho-
`bic substrate-binding pocket include Val80, Leu83, Ile106,
`Gly107, and Phe171 (Figs. 1A, 2A), with Val80 and Leu83
`being well positioned to interact with the propanoic acid
`methyl group. The terminal carbon atom (CZ) of the Phe171
`side chain is 4.1 A˚
`from the phenoxy group of (R)-
`mecoprop; since LigPlot has a 4.0 A˚ threshold for hydro-
`phobic interactions, this interaction is missed in Figure 2A.
`To directly test the importance of potential substrate-
`binding residues of RdpA identified by the homology
`modeling and substrate docking procedures, variant forms
`of the enzymes were created by site-directed mutagene-
`sis. To eliminate the bulky and polar Tyr221 and Arg285
`residues, Y221A and R285A mutants were generated. In
`the presumed ‘‘hydrophobic pocket,’’ Val80, Leu83, Ile106,
`and Phe171 each were changed to alanine to reduce hy-
`drophobic interactions and thereby decrease the binding
`
`Figure 1. Stereo views of the most favorable models of the active sites of RdpA (A) and SdpA (B) with bound substrates. Shown are
`selected protein residues that are predicted to interact with the substrate. For clarity reasons, the FeII ligands are not depicted. (R)- and
`(S)-mecoprop are indicated in light blue and in turquoise, respectively, and aKG in yellow.
`
`www.proteinscience.org
`
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`Fig. 1 live 4/C
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`Mu¨ ller et al.
`
`The specific activities of the RdpA variants were tested
`using (R)-mecoprop and the racemic mixture (Table 1).
`The (S) enantiomer is not sold commercially and was
`available in very limited supply, so the RdpA variants were
`not tested with this compound. When the RdpA variants
`were examined using 4 mM (R)-mecoprop, the V80A and
`F171A variants exhibited ;60% of wild-type enzyme ac-
`tivity; I106A had ;30% of that activity and L83A, Y221A,
`and R285A were ;10% active. The activities of the double
`mutants I106G/G107I and I106G/G107N were further
`reduced compared with that of I106A, consistent with a
`bulkier side chain at position 107 presenting steric hindrance
`to substrate binding in a reactive conformation.
`The RdpA variants retaining at least 10% of wild-type
`enzyme activity were subjected to more detailed kinetic
`characterization (Table 2). The maximal concentration of
`substrates that could be tested was 4 mM due to solubility
`limitations; therefore, Km values higher than 800 mM are
`only approximations and possess large errors. The Km
`values of V80A and R285A RdpA variants were at least
`fivefold increased over that of the wild-type enzyme,
`whereas that of the I106A RdpA variant was more than
`20-fold greater, supporting the described docking orien-
`tation of the model. The effect on the R285A variant can
`be understood in terms of decreased interaction with the
`substrate carboxylate, while the changes observed for the
`V80A and I106A variants are likely to arise from loss of
`hydrophobic interactions. The F171A protein had a three-
`fold lower Km, indicating either that this residue does
`not interact specifically with the phenoxy ring of (R)-
`mecoprop or that F171 is actually slightly hindering (R)-
`mecoprop binding. The calculated kcat of all mutant proteins
`was similar to the wild-type value with the exception of the
`R285A mutant enzyme. Arg285 is postulated to interact
`with both the mecoprop carboxylate and the C-1 carbox-
`ylate of aKG, so it could directly influence catalysis.
`
`Binding of (S)-mecoprop to SdpA
`
`The most favorable orientation of (S)-mecoprop was ob-
`tained with aKG in the twist conformation in SdpA and
`(S)-mecoprop bound as illustrated in Figure 1B (with the
`corresponding ligand interactions shown in Fig. 2B). In
`this model, the substrate carboxylate interacts with the amide
`nitrogen of Ser105, the hydroxyl group of Tyr107, and the
`guanidino nitrogens of Arg274. Additional active site resi-
`dues near the polar carboxylate include His272 and His208.
`Also of
`interest,
`the substrate ether oxygen atom is
`predicted to lie within 3.4 A˚ of the two carboxyl oxygens
`of Glu69. If protonated or bridged by the proton of a bound
`water molecule, the Glu69 carboxyl group could confer
`specificity to the (S) enantiomer by making a hydrogen
`bond with the ether oxygen (a prediction not borne out by
`experimental results, vide infra). Residues predicted to be
`
`Figure 2. LIGPLOT diagrams of the active site models of RdpA (A) and
`SdpA (B). Shown are the most important interactions between the substrate
`and the active site according to the model.
`
`affinity of (R)-mecoprop to the active site. Replacing
`Gly107—positioned near and coplanar with the phenoxy
`ring—with a bulkier hydrophobic residue is expected to
`hinder substrate binding, so this residue was mutated to Ile
`and Asn in the double mutants I106G/G107I and I106G/
`G107N, respectively.
`
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`Dichlorprop hydroxylase enantiospecificity
`
`Table 1. Activity measurements of native and variant forms of RdpA and SdpA
`
`(R)-mecoprop (4 mM)
`
`(R)-mecoprop (2 mM)
`
`(R,S)-mecoprop (4 mM)
`
`Hypothesis
`probeda
`
`Specific activity
`(U/mg protein)
`
`—
`I
`I, II
`II
`I, II
`I, II
`II
`I
`I, II
`I, II
`I, II
`
`7.11 6 1.34
`4.3 6 2.1
`0.37 6 0.09
`4.3 6 0.7
`2.14 6 0.025
`4.83 6 0.31
`5.2 6 0.4
`0.37 6 0.17
`1.1 6 0.1
`0.05 6 0.03
`0.62 6 0.06
`
`(%)
`
`100
`60.5
`5.2
`60.5
`30.1
`67.9
`73.1
`5.2
`14.8
`0.7
`8.7
`
`Specific activity
`(U/mg protein)
`
`5.60 6 0.26
`2.06 6 0.48
`0.24 6 0.06
`3.27 6 0.23
`1.41 6 0.29
`4.75 6 0.22
`4.86 6 0.52
`0.43 6 0.02
`0.92 6 0.06
`0.04 6 0.03
`0.56 6 0.05
`
`(%)
`
`100
`36.8
`4.3
`58.3
`25.2
`84.8
`86.8
`7.7
`16.4
`0.7
`10.0
`
`Specific activity
`(U/mg protein)
`
`6.46 6 0.44
`2.7 6 0.5
`0.27 6 0.02
`2.27 6 0.15
`1.10 6 0.11
`4.59 6 0.35
`4.49 6 0.31
`0.31 6 0.01
`0.80 6 0.06
`0.05 6 0.05
`0.53 6 0.04
`
`(%)
`
`100
`41.8
`4.2
`35.2
`17.0
`71.1
`69.5
`4.8
`12.4
`0.8
`8.2
`
`(S)-mecoprop (4 mM)
`
`(S)-mecoprop (2 mM)
`
`(R,S)-mecoprop (4 mM)
`
`Hypothesis
`probeda
`
`Specific activity
`(U/mg protein)
`
`—
`I, II
`I, II
`I, II
`II
`I, II
`I
`I
`II
`II
`
`25.5 6 1.7
`7.85 6 1.65
`1.22 6 0.3
`4.4 6 0.57
`0.71 6 0.07
`0.35 6 0.1
`0.3 6 0.16
`0.01 6 0.01
`0.07 6 0.03
`0.96 6 0.27
`
`(%)
`
`100.0
`30.8
`4.8
`17.3
`2.8
`1.4
`1.2
`0.0
`0.3
`3.8
`
`Specific activity
`(U/mg protein)
`
`28.96 6 3.6
`6.6 6 1.3
`0.87 6 0.15
`3.13 6 0.52
`0.52 6 0.05
`0.27 6 0.07
`0.27 6 0.16
`0.01 6 0.01
`0.06 6 0.01
`0.93 6 0.26
`
`(%)
`
`100
`22.8
`3.0
`10.8
`1.8
`0.9
`0.9
`0.0
`0.2
`3.2
`
`Specific activity
`(U/mg protein)
`
`23.5 6 0.74
`5.54 6 1.24
`0.53 6 0.08
`2.08 6 0.3
`0.35 6 0.04
`0.14 6 0.05
`0.25 6 0.09
`0
`0.02 6 0.03
`0.83 6 0.26
`
`(%)
`
`100.0
`23.6
`2.3
`8.9
`1.5
`0.6
`1.1
`0.0
`0.1
`3.5
`
`(R)-mecoprop (4 mM)
`Specific activity
`(U/mg protein)
`
`0
`0.51 6 0.22
`0
`0.1 6 0.02
`0.01 6 0.004
`0
`0
`0
`0
`0.01 6 0.01
`
`RdpA Sample
`
`Wild type
`V80Ab
`L83Ac
`Q93Ab
`I106Ac
`F171Ac
`F171Qc
`Y221Ab
`R285Ac
`I106G/G107Id
`I106G/G107Nd
`
`SdpA Sample
`
`Wild type
`E69Ad
`Q162Fe
`R207Ae
`R207Ve
`H208Ad
`H272Ad
`R274Ad
`G97N/N98Gd
`G97I/N98Gd
`
`a The letters indicate whether the mutant was used to probe (I) the predicted binding mode of the substrate or (II) the enantiospecificity of the enzyme.
`b Values were determined by measuring activities of samples isolated from three independent cell cultures.
`c Values were determined by triplicate measurements of a single sample.
`d Values were determined by measuring activities of samples isolated from two independent cell cultures.
`e Values were determined by triplicate (for (R,S)-mecoprop) or duplicate (for (S)- and (R)-mecoprop) measurements of a single sample.
`
`in contact with the hydrophobic ring of (S)-mecoprop in-
`clude Ala71, Ala72, Leu82, Val84, Gly97, Asn98, Gln162,
`and Arg207 (with the latter four residues depicted in Fig.
`1B). p-Cation interactions between positively charged side
`chains like Arg207 and aromatic side groups like that in
`mecoprop can contribute very favorably to ligand binding
`(Mitchell et al. 1994).
`To experimentally test the importance of several of
`these residues for the binding of (S)-mecoprop in the
`active site of SdpA, a series of mutant proteins was con-
`structed. Glu69, His208, His272, and Arg274 each were
`changed to alanine to eliminate the bulky and charged
`side chains proposed to interact with the substrate carbox-
`ylate. The extended side chain of Arg207 was eliminated
`in the R207A SdpA variant, and Gln162 was substituted
`with the corresponding hydrophobic residue found in
`RdpA to generate the Q162F variant.
`The specific activities of the SdpA variants were tested
`using the (S) enantiomer, the racemic mixture, and the (R)
`enantiomer of mecoprop (Table 1). The activity of all
`SdpA mutants was strongly reduced. The most active
`
`SdpA variants were the E69A and R207A proteins, while
`all other variants exhibited <5% of the wild-type enzyme
`activity. No activity was detected in the R274A sample.
`E69A, R207A, and H208A were subjected to detailed
`kinetic characterization (Table 2). When analyzed with
`(S)-mecoprop, the R207A and H208A samples had 15-
`and 18-fold increases, respectively, in their Km values
`compared with the wild-type enzyme. The H208A result
`is compatible with an ionic interaction between the mecoprop
`carboxylate group and His208. A similar interaction might
`be possible with the highly flexible Arg207 residue, or
`this side chain may participate in a p-cation interaction
`with the aromatic group of the substrate, in either case
`accounting for the large Km change in the R207A variant.
`In contrast to these results, the E69A variant exhibited
`a lower Km than the native SdpA, arguing against the
`ether oxygen having a significant
`role in substrate
`binding. With (S)-mecoprop, the kcat was reduced four-
`fold for the E69A and R207A SdpA mutants compared
`with wild-type enzyme, and even greater reductions were
`noted for the H208A variant.
`
`www.proteinscience.org
`
`1361
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`
`PGR2023-00022 Page 00006
`
`

`

`JOBNAME: PROSCI 15#6 2006 PAGE: 7 OUTPUT: Friday May 12 01:28:48 2006
`csh/PROSCI/111782/ps0520594
`
`Mu¨ ller et al.
`
`Table 2. Kinetic parameters of selected variant forms of RdpA and SdpA
`
`RdpA
`Variant
`
`Wild type
`V80Aa
`Q93Aa
`I106Ab
`F171Ab
`F171Qb
`R285Aa
`
`(R)-mecoprop
`
`Range (mM)
`
`100–4000
`200–4000
`200–4000
`500–4000
`200–4000
`62.5–4000
`200–4000
`
`Km (mM)
`
`380 6 28
`2300 6 1100
`2800 6 400
`8900 6 2100
`108 6 7
`438 6 34
`1550 6 220
`
`ÿ1)
`kcat (min
`
`252 6 5
`248 6 56
`260 6 21
`238 6 42
`175 6 2
`203 6 4
`52 6 4
`
`ÿ1
`ÿ1)
`kcat/Km (min
`*mM
`
`0.66
`0.11
`0.09
`0.03
`1.62
`0.46
`0.03
`
`(S)-mecoprop
`
`(R,S)-mecoprop
`
`SdpA
`Variant
`
`Wild type
`E69Ac
`R207Ad
`H208Ac
`G97I/N98Gc
`
`Range
`(mM)
`
`31–4000
`31–4000
`250–4000
`500–4000
`250–4000
`
`Km
`(mM)
`
`kcat
`ÿ1)(min
`
`
`kcat/Km
`ÿ1
`ÿ1)
`*mM
`(min
`
`161 6 20
`91 6 14
`2500 6 400
`2900 6 960
`1100 6 270
`
`1010 6 44
`241 6 8
`238 6 20
`21 6 4
`42 6 4.4
`
`6.24
`2.65
`0.1
`0.007
`0.04
`
`Range
`(mM)
`
`31–4000
`222–4000
`250–4000
`N.D.
`N.D.
`
`Km
`(mM)
`
`kcat
`ÿ1)(min
`
`
`kcat/Km
`ÿ1
`ÿ1)
`*mM
`(min
`
`234 6 18
`181 6 38
`2780 6 370
`
`899 6 24
`194 6 8.4
`117 6 8.4
`
`3.85
`1.1
`0.04
`
`N.D., not determined.
`a Values were determined by measuring activities of samples isolated from three independent cell cultures.
`b Values were determined by triplicate measurements of a single sample.
`c Values were determined by measuring activities of samples isolated from two independent cell cultures.
`d Values were determined by triplicate (for (R,S)-mecoprop) or duplicate (for (S)- and (R)-mecoprop) measurements of a single sample.
`
`Residues determining enantiospecificity in RdpA
`and SdpA
`
`A direct comparison of the active sites in the RdpA and
`SdpA models is shown in Figure 3. The different substrate
`enantiomers are predicted to bind with remarkably similar
`geometries in the two predicted structures, with the
`carboxyl groups and aromatic rings nearly overlapping
`but at distinct angles. RdpA residues Leu83, Gln93, Ile95
`(omitted for clarity), Ile106, Phe171, and Arg285 (shown
`in Fig. 1A) could hinder binding of (S)-mecoprop; the
`corresponding residues in SdpA are Ala72, Leu82, Val84,
`Gly97, Gln162, and His272, respectively. Similarly, SdpA
`residues Glu69, Asn98, Ser161, His208, and Arg207
`could restrict binding of (R)-mecoprop; Val80, Gly107,
`Val170, Tyr221, and Val220 occupy these positions in
`RdpA. Ala72, Gly97, and Gln162 are predicted to allow
`access by the (S) enantiomer. In general, side-chain inter-
`actions suggest that SdpA is less specific than RdpA for
`its substrate. This finding is compatible with activity mea-
`surements (Table 1), such as the activity with 2 mM (S)-
`mecoprop versus that with 4 mM of the racemate, that show
`inhibition of SdpA activity by (R)-mecoprop. However,
`RdpA remains completely active toward the (R) enantiomer
`in the presence of (S)-mecoprop (Mu¨ller 2004).
`Site-directed variants of RdpA and SdpA, created and
`purified as described earlier, were used to test a subset of
`the above-mentioned residues for their importance in con-
`trolling enantioselectivity. Single substitutions included
`
`the L83A, Q93A, I106A, F171A, F171Q, and R285A
`variants of RdpA along with the E69A, Q162F, R207A,
`R207V, and H208A variants of SdpA. In addition, to test
`the possibility that Ile106/Gly107 (large residue/small resi-
`due) in RdpA versus Gly97/Asn98 (small residue/large
`residue) in SdpA confers some measure of enantiospeci-
`ficity to the enzymes, the I106G/G107I and I106G/G107N
`double variants of RdpA along with G97N/N98G and
`G97I/N98G double variants of SdpA were generated.
`Each enzyme was tested for activity using its enantiomer,
`the racemate, and in the case of the SdpA mutants, with
`(R)-mecoprop, too (Tables 1, 2).
`that show
`Among the RdpA variants were several
`evidence of inhibition by the (S) enantiomer. For exam-
`ple, the Q93A variant retains 60% of wild-type enzyme
`activity, but this dropped to 35% when the racemate was
`provided, suggesting that Gln93 prevents the incorrect
`enantiomer from binding to and inhibiting the enzyme.
`Significant decreases in activity also were apparent when
`comparing rates using the racemic mixture of mecoprop
`versus the (R)-enantiomer with the Y221A and R285A
`variants; these results suggest that Tyr221 and Arg285
`contribute to enantiospecificity. These variant proteins
`exhibited ;5% to ;15% of the wild-type enzyme activ-
`ity. Less dramatic differences were observed using the
`I106A, F171A, F171Q, and I106G/G107N variants, but in
`each case the activity was reduced in the presence of the
`incorrect
`enantiomer. The
`I106A, F171A, F171Q,
`and R285A proteins were subjected to detailed kinetic
`
`1362
`
`Protein Science, vol. 15
`
` 1469896x, 2006, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1110/ps.052059406, Wiley Online Library on [27/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`PGR2023-00022 Page 00007
`
`

`

`JOBNAME: PROSCI 15#6 2006 PAGE: 8 OUTPUT: Friday May 12 01:28:48 2006
`csh/PROSCI/111782/ps0520594
`
`Dichlorprop hydroxylase enantiospecificity
`
`Figure 3. Stereo views of the superimposition of the substrate-bound active site models of RdpA and SdpA. Shown are selected
`residues predicted to be involved in dictating enantiospecificity. Amino acid residues of RdpA are depicted in pink; residues of SdpA,
`in green. (R)-mecoprop is shown in purple; (S)-mecoprop, in turquoise. (A) View with the focus on the propanoic acid moiety and its
`binding to the active sites. (B) Same as in A but rotated to focus on the phenoxy ring and its interaction with the amino acid residues
`Ile106/Gly107 in RdpA and Gly97/Asn98 in SdpA.
`
`analysis with (R)-mecoprop (Table 2). With the exception
`of the R285A variant (which exhibited ;20% of the wild-
`type enzyme kcat),
`the proteins retained >70% of the
`wild-type enzyme kcat. The I106A and R285A variants
`exhibited 23- and fourfold increases in Km; the F171Q
`form exhibited a Km similar to the wild-type enzyme, and
`the F171A mutant had a threefold lower Km. The latter
`result is consistent with Phe171 hindering binding of the
`correct (R) enantiomer while helping to exclude the (S)
`enantiomer
`from the RdpA active site, s