Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not

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Streptomyces wadayamensis MppP is a PLP-dependent oxidase, not an oxygenase Lanlan Han, Nemanja Vuksanovic, Sarah A Oehm, Tyler Grant Fenske, Alan W. Schwabacher, and Nicholas Silvaggi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00130 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018

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Biochemistry

Streptomyces wadayamensis MppP is a PLPdependent oxidase, not an oxygenase Lanlan Hana, Nemanja Vuksanovica, Sarah A. Oehma, Tyler G. Fenskea, Alan W. Schwabachera, Nicholas R. Silvaggia* a

Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee,

WI 53201, USA

FUNDING STATEMENT: This work was supported by grant CHE-1606842 from the National Science Foundation, Division of Chemistry.

KEYWORDS: Pyridoxal 5’-phosphate, quinonoid, hydroxylase, enduracididine, nonproteinogenic amino acid, secondary metabolism.

ABBREVIATIONS:

PLP,

pyridoxal

5’-phosphate;

L-End,

L-enduracididine;

MPP,

mannopeptimycin; ATase, aminotransferase; SwMppP, Streptomyces waddayamensis MppP.

ACS Paragon Plus Environment

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Abstract The PLP-dependent L-arginine hydroxylase/deaminase MppP from Streptomyces wadayamensis (SwMppP) is involved in the biosynthesis of L-enduracididine, a nonproteinogenic amino acid found in a number of nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of L-arginine to form a mixture of 2-oxo4(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady state kinetics analysis in the presence and absence of catalase show that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo4(S)-hydroxy-5-guanidinovaleric acid produced, 2 molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either ESI-MS. Incorporation of

[18]

[18]

O2 or H2[18]O and analyzing the products by

O was only observed when the reaction was performed in H2[18]O.

Crystal structures of SwMppP with L-arginine, 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at 2.2 Å, 1.9 Å and 1.8 Å resolution, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of Nterminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of L-arginine.


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Introduction Dioxygen is an unusual molecule in that it has a triplet ground state. As a consequence, despite a large thermodynamic driving force, dioxygen is slow to react with singlet state molecules in the absence of a catalyst. In biological systems, catalysts for reactions involving molecular oxygen are enzymes that use redox-active metal ions and/or organic prosthetic groups that activate dioxygen for reactions with singlet ground state molecules by single electron reduction. The cofactors commonly associated with oxygenases, enzymes that incorporate one or both atoms of dioxygen into their products, include flavins (e.g. FAD, FMN), redox-active metal ions (e.g. Fe(II), Mn(II), Cu(II)), and combinations of organic molecules and metal (e.g. the porphyrin ring of heme, or tetrahydropterins)

1-7

. A cofactor that is not normally associated with oxygenases is

pyridoxal-5’-phosphate (PLP). PLP-dependent enzymes are known to catalyze a wide variety of non-electron

transfer

reactions,

including

transamination,

racemization,

β-

and

γ-

elimination/substitution, deamination, decarboxylation, and isomerization8-13. The central feature common to all of these reactions is the formation of a carbanion intermediate that is stabilized by the PLP, which acts as an “electron sink”

14, 15

. Unlike typical oxygenases, PLP-dependent

enzymes are not generally thought of as stabilizing radical intermediates, except in cases where additional cofactors like an iron-sulfur cluster and/or S-adenosyl methionine are involved 16. However, reactions of the carbanionic intermediate of a PLP-dependent enzyme with molecular oxygen are not completely unknown. DOPA decarboxylase, for example, is known to catalyze oxidative deamination as a side-reaction

17

. Also, phenylacetaldehyde synthase first

decarboxylates phenylalanine, then reacts the quinonoid carbanion with molecular oxygen to ultimately produce phenylacetaldehyde 18. Likewise, a number of “para-catalytic” reactions with O2 have been described in other PLP-dependent decarboxylases

19

. Our report in 2015 that the


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PLP-dependent enzyme MppP from the mannopeptimycin biosynthetic pathway in Streptomyces wadayamensis is an

L-arginine

hydroxylase/deaminase expanded the list of PLP-dependent

enzymes that use dioxygen as a substrate

20

. The following year, Du et al.

21

published their

independent discovery of a remarkably similar enzyme, Ind4 from the indolmycin biosynthetic pathway in Streptomyces griseus. What is most interesting about MppP and Ind4 is that they have apparently evolved to allow the 1-electron reduction of dioxygen and possibly to stabilize one or more radical intermediates without the aid of additional cofactors. Yet each enzyme converts L-arginine into a different 4-electron-oxidized product. Streptomyces wadayamensis MppP (SwMppP) catalyzes the direct oxidation of an unactivated C-C bond to yield 2-oxo-4-hydroxy-guanidinovaleric acid (Scheme 1, 1) to begin the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End, 2).

L-End

or its

derivatives, such as β-hydroxy-L-End are found in a number of antimicrobial peptides like mannopeptimycin 22, 23, enduracidin 24, 25, and teixobactin 26 that are active against drug-resistant pathogens. Our previous study of SwMppP established that the quaternary and tertiary structures are nearly indistinguishable from those of the typical fold type I aminotransferases like aspartate aminotransferase (AAT)

27

. The active sites of SwMppP and AAT are, likewise, very similar

with only a limited number of substitutions at key positions, such as His29 and Asp218 to account for the very different catalytic activities of these two enzymes. In addition to difference in active site residues, the N-terminus of SwMppP was not ordered in the crystal structures of the internal aldimine nor the external aldimine with D-Arg. This is somewhat unusual as the Ntermini of many PLP-dependent enzymes cover the active sites and protect the catalytic centers from solvent. The structure of the complex with D-Arg also showed a likely substrate binding mode with the α-carboxylate interacting with Arg352 and the guanidinium group of the side


4

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Biochemistry

chain clamped between two aspartate residues, Asp27 and Asp227, the so-called “aspartate vise.” UV/Vis spectra of SwMppP reacting with L-Arg under aerobic conditions showed the slow accumulation of a peak at 510 nm and a second peak at 560 nm that accumulated until the 510 nm peak reached its maximum, at which point the 560 nm species decayed. These two features were proposed to be quinonoid intermediates and formed the basis for our first proposed mechanism (see Scheme 2 in Han, et al.

20

). This mechanistic hypothesis explained the spectra

and products observed, but several predictions remained to be tested. First, the mechanism predicted the release of ammonia as a product. Second, we hypothesized that the hydroxyl oxygen atom derived from dioxygen, and thus that only one dioxygen molecule would be consumed in each catalytic cycle. As a consequence of this assumption, our hypothesis also predicts that H2O2 would only be released during uncoupled turnover resulting in the abortive product 2-oxo-5-guanidinovaleric acid (3). In addition, the structure with D-Arg presented a very plausible binding site for the side chain, but it was unclear whether or not the actual substrate, LArg, would also bind in the “aspartate vise.” Scheme 1

Here we present experiments to detect the production of ammonia, estimate the stoichiometry of dioxygen consumption, and determine the origin of the hydroxyl oxygen atom of the fully


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oxidized product (1). In addition, the structures of SwMppP in complexes with L-Arg, as well as each of the products have been determined in order to determine if the “aspartate vise” is relevant to the binding of the true substrate. The structure of SwMppP with 1 bound suggests the stereospecificity of the hydroxylation step. The structure with L-Arg bound as the external aldimine suggested that residues at the N-terminus might be important in terms of substrate binding and/or catalysis. We present steady state kinetics on several N-terminal point mutants, as well as a truncation mutant where residues 1-22 have been eliminated. A fourth crystal structure of the SwMppPE15A point mutant with L-Arg bound indicates that the effects of these mutations are more subtle than altering substrate binding or interfering with a specific chemical step.

Materials and Methods Cloning, Expression, and Purification of wild-type and mutant SwMppP. Wild-type SwMppP was cloned previously 20 and was expressed and purified using the published procedure. The four mutants, SwMppPT12A, SwMppPE15A, SwMppPE15Q, and SwMppPT12A/E15A were obtained using the Q5 site-directed mutagenesis kit (New England Biolabs Inc., Ipswich, MA) using freshly purified pE-SUMOkan-SwMppP expression plasmid as the template. The primers for constructing the

T12A

mutant

were:

5’-CTGAAAGAAAACCTGGCGCAATGGG-3’

and

5’-

CCCATTGCGCCAGGTTTTCTTTCAG-3’. The E15A and E15Q variants shared the same reverse primer: 5’-AGGTTTTCTTTCAGTTGCGGTTG-3’; the forward primers were 5’GACGCAATGGGCATACCTGGCAC-3’

and

5’-GACGCAATGGCAATACCTGGCAC-3’,

respectively. The T12A/E15A double mutant was obtained using the primers for the T12A single mutant with the pSUMO-SwMppPE15A plasmid as the template. The N-terminal truncation mutant SwMppP∆1-22 was subcloned into the pE-SUMOkan expression vector (LifeSensors Inc.,


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Biochemistry

Malvern,

PA)

using

primers

containing

BsaI

and

TAGGTCTCTAGGTCTGAACATCGCAGACGGC-3’

XbaI and

restriction 5’

sites

(5’-

GCTCTAGATCATTAGCGGGTTTCCAGGAC-3’). All five mutants were expressed and purified as described for the wild-type protein

20

. The proteins were desalted using a 2 x 5 mL

HiTrap desalting column (GE Lifesciences) into 10.0 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 µM PLP, pH 6.7 and stored at −80°C.

Crystallization of SwMppP with substrate and products bound. SwMppP crystals were obtained by the method described previously 20. Briefly, 1-2 µl of 15 mg/ml SwMppP in 10 mM MES, 20 µM PLP, pH 6.7 were mixed with 1 µl of 30 % polyethylene glycol monomethyl ether 550 (PEG MME 550), 50 mM MgCl2, and 0.1M HEPES pH 8.0. The structure of SwMppP with L-Arg

bound was obtained by transferring crystals into 30 µL drops of the crystallization solution

supplemented with 10 mM L-Arg and 20% glycerol. After being soaked for 5 h at room temperature, crystals were flash-cooled by being plunged into liquid nitrogen. The structure of SwMppP with 1 bound was obtained by transferring crystals into 30 µL drops of the crystallization solution supplemented with a nominal 5 mM concentration of reaction products. This was prepared by incubating SwMppP (10 µM) and catalase (100 ng/ml) with 2 mM L-Arg, pH 9.0, in MilliQ water (5 mL total volume) overnight at room temperature. The solvent containing the reaction products was evaporated to dryness in a CentriFan PE evaporator (KD Scientific Inc., Holliston, MA). The resulting solid was made up to a nominal 20 mM concentration (assuming that all of the material present was 1), which was used to assemble soaking solutions with a nominal 5 mM 1. After being soaked for 24 h at 22 oC, crystals were cryo-protected by sequential soaks in holding solutions containing 10, or 20% glycerol and flash-


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cooled by being plunged into liquid nitrogen. The structure of SwMppP with 3 bound was obtained by transferring crystals into 30 µL drops of the crystallization solution supplemented with 5 mM 2-oxo-5-guanidinovaleric acid prepared using rattlesnake L-amino acid oxidase as described

28

. After being soaked for 24 h at 22 oC, crystals were cryo-protected by paratone N

and flash-cooled by plunging in liquid nitrogen.

Data collection and Structure solution. X-ray diffraction data for the SwMppP·L-Arg complex were collected at beamline 21-ID-F of the Life Science Collaborative Access Team (LS-CAT) at the Advanced Photon Source (APS). The SwMppP·1 and SwMppP·3 data sets were collected at LS-CAT beamline 21-ID-D. The SwMppP·L-Arg and SwMppP·1 data were processed with HKL2000 29. The SwMppP·3 data was processed using MOSFLM 30. The molecular replacement solutions were obtained using PHASER

31

as implemented in

CCP4 32 using the unliganded SwMppP dimer (with PLP, solvent, and H atoms removed, and all B-factors reset to 20.0 Å2) as the search model (PDB ID 5DJ1

20

). Maximum likelihood-based

refinement was performed using the phenix.refine33 program implemented in the PHENIX suite 34

. The models underwent iterative rounds of building in COOT

35

followed by refinement in

phenix.refine. Ordered solvent molecules were added automatically in phenix.refine and culled manually in COOT. Hydrogen atoms were added to the model using phenix.reduce

36

and were

included in the later stages of refinement to improve the stereochemistry of the model. Positions of H atoms were refined using the riding model with a global B factor. Regions of the model for translation-libration-screw (TLS) refinement were identified using phenix.find_tls_groups (P. V. Afonine, unpublished work), and the TLS parameters were refined in phenix.refine. Once the refinements converged, the models were validated using the tools implemented in COOT and


8

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Biochemistry

PHENIX. Sections of the backbone with missing or uninterpretable electron density were not included in the final models. Side chains with poor or missing electron density were modeled in favored rotameric conformations, and the B factors were allowed to refine without additional restraints. This was done to alert end users that these residues are not well-defined in the electron density. Restraints for the phenix.elbow

37

L-Arg-PLP

external aldimine, 1, and 3 were generated with

. Data collection and model refinement statistics are listed in Table 1.

Coordinates and structure factors for all four SwMppP models described here have been deposited in the Protein Data Bank as entries 6C8T, 6C9B, 6C92 and 5BK7.

Nuclear magnetic resonance analysis of reaction products. All reactions (4.8 mL) were performed in 50 mL conical tubes with slow agitation to maintain dioxygen saturation. All enzymes were desalted into 20 mM sodium phosphate, 10 µM PLP, pH 8.4 and concentrated to ~600 µM to minimize the amount of free PLP added to the reaction mixtures. For the wild-type SwMppP, reactions contained ~10 µM enzyme and 2 mM L-Arg in 20 mM sodium phosphate, pH 8.4. Due to the lower efficiency of the mutants, these reactions contained ~65 µM enzyme. Control reactions of 2 mM L-Arg in 20 mM sodium phosphate, pH 8.4 without enzyme were also analyzed. All reactions were incubated at 25 oC, 200 rpm for 2.5 h. Owing to light sensitivity of the enzymes, reactions were kept dark. After the incubation period, reactions were evaporated to dryness using the CentriFan PE evaporator at 40 oC for 6 h. The resulting solid was taken up in 600 µL D2O and centrifuged at 13000 x g for 10 min to remove undissolved precipitate (presumably insoluble, aggregated enzyme). The 1H NMR spectra were recorded on a DRX-500 MHz spectrometer (Bruker) equipped with a broadband (BBO) probe.


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Mass spectroscopy analysis of SwMppP reaction products. Because mass spectrometry is more sensitive than NMR, the products of SwMppP and the mutants were also analyzed by mass spectrometry. All reactions (1.0 mL) were conducted in 15 mL conical tubes. To remove as many confounding solutes as possible, the enzymes used for these experiments were desalted into MilliQ water immediately prior to assembling the reactions. The reactions contained 2 mM L-Arg

in MilliQ H2O, 10 µM enzyme in MilliQ H2O, with 53 U catalase from bovine liver

(Sigma-Aldrich, St. Louis, MO). The catalase was added to prevent the peroxide-mediated decarboxylation of the ketoacid products of SwMppP. Samples containing only 2 mM L-Arg (with or without catalase) served as negative controls. After incubating for 2.5 h at 25°C, 200 rpm shaking, all samples were boiled for 10 min and centrifuged at 30,000 x g for 10 min to remove precipitated protein and any other insoluble material. The supernatants were diluted 10fold with MilliQ H2O and were analyzed by direct injection into an LC-MS 2020 Single Quadrupole Liquid Chromatograph Mass Spectrometer (Shimadzu).

Steady State Enzyme kinetics. All 1.0 mL kinetic assays were conducted in triplicate at 25 °C in 50 mM BIS-TRIS propane, pH 9.0. The initial velocity of SwMppP-catalyzed oxidation of LArg was measured directly by monitoring the decrease in the dioxygen concentration using a Clark type electrode (Hansatech Instruments, Norfolk, U.K.). The concentration of L-Arg was varied from 10 to 10,000 µM. The kcat and KM values were determined from the initial velocity data using the equation v0 = V[S]/(KM + [S]), where [S] is the concentration of L-Arg, v0 is the initial velocity, V is the maximal velocity, and KM is the Michaelis constant. These reactions were also used to determine the stoichiometry of dioxygen consumption. The reactions were allowed to plateau, and the dioxygen concentration at this point was subtracted from the initial


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Biochemistry

concentration of dioxygen to determine the amount of O2 consumed in the reaction. The ratio of dioxygen consumed to the concentration of L-Arg in the reaction was taken as a rough indicator of stoichiometry, since the ammonia detection assay (see below) suggested that, at concentrations of L-Arg below the dioxygen concentration of air-saturated buffer (~250 µM), approximately 70% of the L-Arg is consumed by SwMppP.

Confirming production of H2O2 and ammonia by SwMppP. To establish whether or not H2O2 was a product of the MppP-catalyzed oxidation of L-Arg, the regeneration of O2 by catalase was measured using the Clark type oxygen electrode. Triplicate reactions (1.0 mL) containing 25, 50, or 100 µM L-Arg in 50 mM BisTris propane, pH 9.0 were initiated with the addition of 10 µM SwMppP. When the reactions reached equilibrium, 5 µg/mL catalase was added and the resulting increase in the dioxygen concentration was recorded. The production of ammonia was confirmed using a glutamate dehydrogenase (GDH)-coupled assay as described

38

. Triplicate reactions (1.0 mL) were conducted in 50 mM HEPES, pH 7.5

and included 20 µM SwMppP, 20 or 50 µM L-Arg, 140 µM NADH, and 3.5 mM α-ketoglutarate. The reaction components were mixed and incubated for 10 min at room temperature. The equilibrated reaction mixture was used to blank the spectrophotometer and the absorbance at 340 nm was monitored to ensure a flat baseline. The dehydrogenase reaction was initiated by the addition of 10-2 units of GDH (Sigma) and the absorbance at 340 nm was recorded until the reaction reached equilibrium. The change in absorbance at 340 nm was compared to a standard curve to estimate the concentration of ammonia consumed.


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Isotopic labeling to define the origin of the hydroxyl oxygen atom of 1. Reactions in the presence of 18O2 were conducted in a closed airtight reaction system. The tonometer containing 5 mL of 10 µM SwMppP in 100 mM ammonium bicarbonate, pH 9.0 with 50 ng/mL catalase in the body, and 20 µL of 500 mM L-Arg in one arm was made anaerobic by 45 cycles of vacuum followed by purging (~30 s) with argon gas. The other arm of the tonometer was fitted with a gas-tight connection to a small lecture bottle of 18O2. After the final argon purge, the atmosphere in the tonometer was removed to create a vacuum. The enzyme and substrate solutions were then mixed by gently inverting the tonometer several times. The yellow-green MppP solution immediately turned pink, indicating that the enzyme had reacted with L-Arg and was arrested due to lack of dioxygen. The valve between the tonometer and the [18]O2 cylinder was opened and the contents of the tonometer were gently rocked to promote oxygenation of the reaction mixture. The tonometer stood for 2 h at room temperature in the dark. The reaction mixture was analyzed by direct injection into the LC-2020 ESI-MS. The reactions in H2[18]O contained 10 µM SwMppP, 2 mM L-Arg, and ~95% H2[18]O in the same ammonium bicarbonate buffer (200 µL total volume). These were assembled in 1.7 mL microcentrifuge tubes and incubated overnight at room temperature, in the dark. The reaction products were analyzed directly by ESI-MS.

Results and Discussion Structure of the SwMppP·L-Arg complex. As alluded to above, the structures of the internal aldimine and

D-Arg-bound

forms of SwMppP show that in the absence of substrate, the N-

terminus of the enzyme (residues 1-22) is either completely disordered or makes interactions with a neighboring molecule in the crystal 20. In either case, the PLP cofactor is left exposed to solvent. By soaking crystals of wild-type SwMppP in a solution containing 10 mM L-Arg, we


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Biochemistry

were able to trap the enzyme-substrate complex. There was no change in the unit cell dimensions or crystal packing as a result of

L-Arg

binding; the asymmetric unit still contained four

protomers arranged as two independent dimers. Superimposing the L-Arg-bound structure on that of the internal aldimine (i.e. holoenzyme) using Secondary Structure Matching (SSM)

39

shows

that there is no domain movement upon substrate binding (root mean square deviation for matched Cα atoms is 0.4 Å), as has been observed for some PLP-dependent aminotransferases 40. Indeed, the only differences between the holoenzyme and

L-Arg-bound

structures are the

position of the N-terminus, which is ordered in the SwMppP·L-Arg structure (Figure 1), and the presence of L-Arg in the active site, where it is bound to PLP as the external aldimine (Figure 2). The N-terminus in the SwMppP·L-Arg complex is disordered to Lys8 (the first residue visible in the electron density map) and forms a short α-helix (residues 11-20) that serves to isolate the catalytic center from the bulk solvent. The side chains of Thr12 and Glu15 interact with the guanidinium and α-carboxylate groups of the substrate, respectively (Figure 2). These direct interactions with the substrate prompted us to examine the role of the N-terminus in more detail (see below). As observed in the structure of the external aldimine with D-Arg (PDB ID 5DJ3) 20, the α-carboxylate of L-Arg makes a salt bridge interaction with Arg352. Unlike D-Arg, however, L-Arg

binds such that the guanidinium group interacts primarily with the phosphate group of the

cofactor, and not with the “aspartate vise.” This was not unexpected; since

D-Arg

is a

competitive inhibitor of the reaction and not a substrate, it is not surprising that the two enantiomers would have different binding modes. The guanidinium group of

L-Arg

also

participates in hydrogen bonding interactions with Ser91 and the main chain carbonyl groups of Ser248* and Asp249* from the other protomer of the dimer.


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Comparing the structures of the SwMppP internal aldimine and the external aldimine with LArg, there are only a few significant changes in the orientations of active site residues (Figure 3). The plane of the PLP ring pivots upward from the pyridine N atom, which is anchored by its interaction with Asp188. The phenyl ring of Phe191 rotates approximately 90°, likely to partially fill the void created by the movement of the PLP ring. Formation of the external aldimine also occasions a re-orientation of Lys221 to make either a direct or water-mediated hydrogen bonding interaction with Asp218, as well as a potential hydrogen bond to the PLP phosphate group. Asp218 is conserved among MppP homologs and may play a role in discouraging premature hydrolysis of the external aldimine 20. This is a similar set of interactions to those observed for Lys221 in the external aldimine with D-Arg. The side chain of His29, which adopts multiple conformations in the internal aldimine is stabilized in a single conformation in the external aldimine, though it makes no discernable interactions with the enzyme, substrate, or cofactor. Finally, the ring of Phe115 is displaced by the movement of the cofactor and comes into van der Waals contact with the aliphatic portion of the L-Arg side chain.


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Biochemistry

Figure 1. The L-Arg-bound SwMppP dimer with one protomer shown in cartoon representation, and the other as a solvent-accessible surface (A). The large and small domains are colored blue and green, respectively. The N- and C-termini are labeled. The white arrow and yellow sphere denote the position of Leu23, the first residue visible in the internal aldimine structure (PDB ID 5DJ1 20). Upon substrate binding, the N-terminus orders into a short α-helix (purple) that covers the catalytic center and sequesters it from bulk solvent. A stereoview of the N-terminus shows the electron density for this portion of the structure (B). The yellow sphere indicates the position of Leu23. None of the section in purple is visible in the unliganded or D-Arg-bound structures of the enzyme. The 2|Fo| - |Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo| - |Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. This figure was prepared using the POVSCRIPT+ modification of MOLSCRIPT41, 42

and POV-Ray (www.povray.org).


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6C9B

SwMppP·2KA 6C92

SwMppPE15A·Arg 5BK7

P212121

P212121

P212121

P21

a, b, c (Å)

85.8, 108.4, 196.2

86.0, 108.4, 196.5

85.9, 108.8, 195.5

64.4, 200.5, 139.9

α, β, γ (°)

90, 90, 90

90, 90, 90

90, 90, 90

90, 103.3, 90

50.00-2.20 (2.24-2.20)

50.00-1.93 (1.96-1.93)

44.59-1.83 (1.87-1.83)

50.00-2.20 (2.28-2.24)

0.97872

0.97856

0.97849

408941 (15090)

650654 (25173)

1055413 (53434)

977686 (170041)

85820 (3839)

135654 (6682)

160466 (7902)

36991 (8030)

91.6 (84.1)

98.4 (98.3)

99.9 (100.0)

97.0 (92.2)

0.057 (0.193)

0.066 (0.488)

0.074 (0.567)

0.110 (0.588)

4.8 (3.9)

4.8 (3.8)

6.6 (6.8)

5.7 (4.6)

24.7 (6.1)

21.8 (2.3)

14.4 (3.3)

14.9 (2.1)

160326 2000 0.151 (0.175) 1434 1032 31

156239 2001 0.205 (0.266) 2839 1432 72

31.2

46.5

SwMppP·4HKA

PDB ID

SwMppP·Arg 6C8T

Space group Cell dimensions

Resolution (Å) (last shell)

a

Wavelength (Å) No. of reflections Observed Unique Completeness (%) Rmerge (%)

a

a,b

Multiplicity

a

Reflections in work set Reflections in test set Rcryst (Rfree) No. of residues No. of solvent atoms Number of TLS groups

85724 1999 0.145 (0.195) 1458 745 30

Model Refinement Statistics 135549 2000 0.156 (0.178) 1454 930 20

Average B-factor (Å2) c 28.8

Protein atoms

32.4 d

d

d

38.0d

Ligands

31.6

Solvent

33.0

36.9

37.3

39.0

Bond lengths (Å)

0.010

0.011

0.010

0.012

Bond angles (°)

1.14

1.16

1.14

1.26

Coordinate error (Å)

0.17

0.19

0.19

0.29

97.2 / 2.5 / 0.3

98.2/ 1.8 / 0

97.6/ 2.0 / 0.4

97.2 / 2.5 / 0.3

41.9

37.6

RMS deviations

Ramachandran

statistics

(favored/allowed/outliers)

Table 1: Crystallographic data collection and refinement statistics.

a

Values in parentheses apply to the high-resolution shell indicated in the resolution row. R = Σ(||Fobs|-scale*|Fcalc||) / Σ |Fobs|. c Isotropic-equivalent B factors, including contribution from TLS refinement. d In the L-Arg complex structures, “Ligands” refers to the L-Arg-PLP unit; in the 2KA complex structure, it refers to the 2KA molecules; in the 4HKA complex structure, it refers to the 4HKA molecules. b


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Biochemistry

Figure 2. Stereoview of the SwMppP active site with the substrate, L-Arg, bound as the external aldimine (A). The 2|Fo| - |Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo| - |Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. The Cα trace and carbon atoms of the main protomer (that containing the catalytic Lys221) are colored light blue, and those of the other protomer in the dimer are colored light green. Residue labels with an asterisk (*) also denote the other protomer. The catalytic Lys221


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Page 18 of 48

and the PLP cofactor are shown as sticks with orange carbon atoms. L-Arg is also shown as sticks, but with light purple carbon atoms. Selected solvent molecules are shown as transparent blue spheres. This figure was prepared using the POVSCRIPT+ modification of MOLSCRIPT41, 42

and POV-Ray. The 3-dimensional figures are reduced to schematic form (B) in order to

highlight relevant interactions and their distances (Å). Hydrogen bonds and electrostatic interactions are shown as green dotted lines, and distances that do not correspond to any particular noncovalent interaction are shown as grey dotted lines. Panel B and subsequent schematics were created with MarvinSketch (ChemAxon) and Adobe Illustrator CC 2017.

Structures of SwMppPproduct complexes. The structures of the SwMppP•1 complex (Figure 4) and the SwMppP•3 complex (Figure 5) both have the products bound noncovalently in positions very similar to that of L-Arg in the external aldimine structure. In neither case did binding of the products perturb the unit cell dimensions or induce shifts in the quaternary or tertiary structure of the enzyme. In the SwMppP·1 structure, three of the four active sites have 1 bound, the other one is empty. Thus, despite the modest excess of 1 over 3, 1 is able to outcompete 3 for binding to the enzyme under the conditions used for crystallization. Similarly, in the SwMppP·3 structure, three active sites have 3 bound, and the fourth is empty. In one of the active sites containing 3, the electron density clearly shows that 3 is bound as the external aldimine, which can only be explained by L-Arg starting material remaining in the preparation of 3 (~10% based on 1H NMR spectra). Both products have been modeled as the ketones, since this is what we have observed by 1H NMR and ESI-MS (see below). It should be noted that the external aldimine might be displaced directly by the attack of Lys221 to release the imine of 1. However this may be, the most important observation from the structure with 1 bound is that the


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Biochemistry

enzyme produces the 4(S)-hydroxy product. The 4-hydroxy group interacts with both the PLP phosphate group and Nε of His29. This arrangement suggests that His29, which is also absolutely conserved among MppP homologs, likely has some role in the hydroxylation (or dehydration in the case of Ind4) at C4 of the arginine side chain. Whether it acts as a general base catalyst, deprotonating H2O, C4, or helps to stabilize the superoxide/peroxyanion species inferred to be involved in the mechanism remains to be determined. The structure with 3 bound is nearly identical to the complex with 1, save for the fact that 3 does not have the 4-hydroxy group. In this sense, the SwMppP·3 complex is likely a good analog of the Michaelis complex with L-Arg.

Figure 3. Stereoview of the internal aldimine form of SwMppP, shown as a grey Cα trace with the internal aldimine (K221-PLP) shown as sticks with yellow carbon atoms, superimposed on the structure of the external aldimine with L-Arg, which is colored as in Figure 2. The chloride ion present in the internal aldimine structure is shown as a green sphere, and water molecules belonging to this structure are shown as transparent yellow spheres. One of these water molecules is displaced by the guanidinium group of L-Arg.


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It is interesting that SwMppP promotes the stereospecific hydroxylation, whereas the homolog Ind4 produces the unsaturated enamine 4, which rapidly tautomerizes to the imine 4’. Whether SwMppP releases its products as the enamine, imine, or ketone is a question that remains to be answered. We have not been able to detect the imine of 1 or 3, but rapid deamination to the corresponding ketones would account for this. Ind4 was shown to release the imine, and that this compound is subject to nonenzymatic hydrolytic deamination to give the ketone from 4, a deadend product. To avoid this, the product of Ind4 (4) is passed to an NADH-dependent reductase, Ind5, that reduces 4 to D-4,5-dehydroarginine and the uncoupled (abortive) product to D-Arg 21. The D-4,5-dehydroarginine is then incorporated into indolmycin, and the D-Arg can be used by other pathways, and is thus not wasted. In the case of SwMppP, the downstream enzyme, MppR, is related to a class of type I (lysine-dependent) aldolases and is able to cyclize 1 to create the iminoimidazolidine ring of L-End 43. Since MppR can react with 1, there is no need to protect the imine, if that is the true product of SwMppP, from nonenzymatic deamination. The 2ketoarginine (3) that is produced by SwMppP is efficiently cycled back to L-Arg by the third enzyme in the pathway, MppQ 20.


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Biochemistry

Figure 4. Stereoview of 1 bound in the active site of SwMppP (A), showing the PLP covalently bound to the enzyme as the internal aldimine. Thr12 and Glu15 interact with the guanidinium and α-carboxylate groups, respectively. His29 interacts with the hydroxyl group of 1. The 2|Fo||Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo|-|Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. The protein and solvent are colored as in Figure 2; the product, 1, is shown as sticks with tan carbon


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Page 22 of 48

atoms. Schematic view of the active site (B) showing potential hydrogen bonding or electrostatic interactions as green, dashed lines.


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Biochemistry

Figure 5. Stereoview of the active site in the SwMppP•3 complex (A), showing the PLP covalently bound to the enzyme as the internal aldimine. As in the complex with 1, Thr12 and Glu15 interact with the guanidinium and α-carboxylate groups of 3, respectively. The 2|Fo|-|Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo|-|Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. Coloring is as in


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Page 24 of 48

Figure 2, except that the carbon atoms of 3 are colored yellow. Schematic view of the active site showing potential hydrogen bonding or electrostatic interactions as green, dashed lines (B).

Stoichiometry of dioxygen consumption and the origin of the hydroxyl oxygen atom of 1. SwMppP reacts with L-Arg and dioxygen to form 1 and 3 in a 1.7:1 ratio. The number of molecules of dioxygen consumed during turnover is a fundamental mechanistic question that was not answered in our first characterization of the enzyme. The proposed mechanism (see

20

)

predicted that only one equivalent of oxygen would be required for the formation of either product, and that only the dioxygen used to create the partially oxidized product 3 would be lost as peroxide. To test these predictions, the amounts of dioxygen consumed in reactions of SwMppP with several concentrations of L-Arg were compared to the amounts of L-Arg added to the reaction (Table 2). Wild-type SwMppP consumed 1.4 equivalents of dioxygen for each equivalent of L-Arg in the reaction. This observation can only be explained by a branched mechanism where one branch requires one equivalent of dioxygen, and the other requires two. To detect the amount of hydrogen peroxide produced, we measured the amount of dioxygen regenerated by the addition of catalase to SwMppP reactions at equilibrium. Catalase invariably produced an amount of dioxygen corresponding to exactly half of what was consumed in the original SwMppP reaction (Table 2, Figure S1). Thus, every molecule of dioxygen used to oxidize L-Arg is reduced to H2O2 by the enzyme.

Table 2. The ratio of the concentration of dioxygen consumed versus the concentration of L-Arg added to reactions of the various forms of SwMppP. Peroxide formation by wild-type SwMppP was also assayed by the addition of catalase. The amount of oxygen generated by catalase is


24

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Biochemistry

always half that consumed by MppP, indicating that each molecule of dioxygen used to oxidize L-Arg

is reduced to H2O2.

[L-Arg] (µM)

SwMppP SwMppP

SwMppP

SwMppP

SwMppP

SwMppP∆1-22

(WT)

(T12A)

(E15A)

(E15Q)

(T12A/E15A)

25

1.40:1

0.64:1

1.05:1

1.44:1

0.76:1

0.61:1

50

1.43:1

0.54:1

0.95:1

1.28:1

0.65:1

0.45:1

100

1.38:1

0.59:1

0.95:1

1.29:1

0.72:1

0.47:1

[O2] consumed by SwMppP (µM)

[O2] generated by catalase (µM)

25

38.1 ± 1.6

19.0 ± 1.0

50

69.3 ± 1.2

35.7 ± 0.6

100

140.3 ± 2.3

71.7 ± 2.1

Given that each molecule of dioxygen consumed by SwMppP is reduced to H2O2, the hydroxyl oxygen atom must necessarily derive from water. To confirm this inference, reactions of SwMppP were run in either

[18]

O2 or H2[18]O and the products were analyzed by ESI-MS.

Catalase was included in all reactions to prevent the peroxide-catalyzed oxidative decarboxylation of 1 and 3

44

. When reactions were run in the presence of

[18]

O2, the [M+H]+

ions were observed at m/z = 174 and 190, corresponding to 3 and 1, respectively (Figure S2). No [18]

O was incorporated into the products. When the reactions in H2[18]O were analyzed by ESI-

MS, the [M+H]+ ions were observed at m/z = 176 and 194. Both the hydroxyl and ketone oxygen atoms, therefore, derive from water. Since dioxygen is not incorporated into the product, SwMppP is an oxidase and not an oxygenase.


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The N-terminus is required for the proper functioning of SwMppP. The observation that the Nterminus of SwMppP becomes ordered upon binding of substrate or products in the active site, together with the direct interactions between Thr12 and Glu15 with the ligands, suggested that the N-terminus may play a role in catalysis. To explore this possibility, a series of SwMppP variants were created and characterized in terms of steady state kinetics and product profiles. The variants tested were T12A, E15A, E15Q, T12A/E15Q, and the truncation mutant SwMppP∆1-22. The effects of all of these mutations on the steady state turnover of SwMppP were very mild. The E15A and E15Q variants were essentially indistinguishable from the wild-type in terms of their steady state kinetic parameters (Table 3), showing a 1.1- and 1.7-fold increase in kcat/KM, respectively. The other two point mutants, T12A and T12A/E15A, exhibited very mild perturbations of the steady state turnover, with kcat/KM values decreased by 15- and 16-fold, respectively, relative to the wild-type SwMppP. Even the SwMppP∆1-22 truncation mutant, which showed the greatest effect, only exhibited a ~10-fold increase in KM and a ~3-fold decrease in kcat amounting to only a 25-fold decrease in kcat/KM.


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Biochemistry

Table 3. Steady state kinetic parameters of SwMppP and mutants as monitored by dioxygen consumption, including the analysis of the products produced by the reaction and, when both products were produced, the relative concentrations of each.

KM,L-Arg (µM)

kcat (s-1)

Product(s) Observeda

kcat/KM,L-Arg (M-1 s-1)

[1]:[3]b

SwMppP

26.50±4.20

0.2025±0.0061

7.6x103 ± 1.2x103

1 and 3

1.7:1

SwMppPT12A

105.80±12.13

0.05112±0.0016

4.8x102 ± 57.42

3

NA

SwMppPE15A

27.99±3.97

0.2358±0.0058

8.4x103 ± 1.2x103

3

NA

SwMppPE15Q

18.95±3.49

0.2376±0.0072

1.3x104 ± 2.3x103

1 and 3

1:2.4

SwMppPT12A/E15A

74.49±7.68

0.03465±0.0019

4.7x102 ± 54.32

3

NA

SwMppP∆1-22

234.80±33.41

0.07185±0.0023

3.1x102 ± 44.63

3 (trace)

NA

a

Products were detected by ESI-MS analysis of reactions run in MilliQ water in the presence of

catalase. b

Relative concentrations were estimated based on the integration of relevant peaks in 1H NMR

spectra (Figure S3).


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Page 28 of 48

Figure 6. Orthogonal views of the external aldimine with L-Arg in the structure of SwMppPE15A, colored as in Figure 2. The Ala15 residue is highlighted with a red label. In all respects, save for the absence of four atoms from the glutamate side chain, this structure almost exactly resembles that of the wild-type SwMppP·L-Arg complex.

As mild as the effects on the steady state kinetics were, it was surprising that all of the mutants, including the E15Q variant, had marked changes in their product profiles. SwMppPT12A, SwMppPE15A, SwMppPT12A/E15A, and SwMppP∆1-22 produced only the partially oxidized product, 3; none of the hydroxylated product was detectable by ESI-MS. The E15Q variant that performed so similarly to the wild-type enzyme in terms of oxygen consumption, produced both 1 and 3, but according to 1H NMR spectra, in a ratio of 1:2.4 (Figure S3). Thus, all of the changes to the N-terminus disrupted the hydroxylation step, shunting catalysis in these variants entirely (or almost entirely in the case of E15Q) down the abortive branch to 3. This is consistent with the idea that the well-ordered state encourages Lys221 to interact with Asp218, rather than prematurely attacking the aldenamine, which leads to the incompletely oxidized product 3.


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Biochemistry

To see if there was a structural explanation for this curious observation, we determined the structure of SwMppPE15A with L-Arg bound. The E15A mutant crystallized in a different space group, P21, with 8 chains (4 independent dimers) in the asymmetric unit. At 2.2 Å resolution, all 8 active sites show clear electron density for the external aldimine with L-Arg (Figure 6). Given the different packing, the structures of SwMppP·L-Arg and SwMppPE15A·L-Arg are remarkably similar (RMSD of 0.34 Å for all Cα atoms). The two structures are superimposable with almost no difference between them save the missing CH2COO- of the Glu15 side chain in the mutant structure. Thus, there is a small void near the α-carbon of the L-Arg substrate in the E15A mutant, but this does not communicate directly with the bulk solvent. It is possible that this pocket accommodates one or more water molecules, even though there are no ordered water molecules visible in the electron density. The simplest explanation for the loss of hydroxylation activity in the N-terminus mutants may be that such voids or the loss of interactions that loosen the grip of the N-terminus on the substrate (e.g. T12A) increase the likelihood that water will intrude in the vicinity of C4’ of PLP during the catalytic cycle, promoting premature hydrolysis of the external aldimine.


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Biochemistry

Figure 7. Revised mechanistic hypothesis consistent with the available experimental observations of SwMppP catalysis. The colored boxes indicate the species that can be detected spectroscopically: I, internal aldimine (415 nm); II, external aldimine (425 nm); Q1, first quinonoid intermediate (510 nm); Q2, second quinonoid intermediate (560 nm). The species in grey boxes are not observed directly.

Refined mechanistic proposal. All of these data have allowed us to refine our initial mechanistic hypothesis for SwMppP. Our new proposed mechanism (Figure 7) takes into account all of the experimental data currently available. The first steps up to the first quinonoid intermediate (Q1) are common steps in PLP-dependent enzymes. The extreme stability of Q1 in the absence of dioxygen (t1/2 of several hours) suggests that the unique active site environment of SwMppP has evolved to stabilize this electron-rich intermediate until it encounters dioxygen. Perhaps the conformational change of the N-terminus on substrate binding allows a well-defined active site with limited access to water. The formation of the superoxide anion and subsequent hydrogen atom abstraction steps at C3 and C4 (IV and V in Figure 7) are speculative, and the same transformation could be accomplished in other ways, for example, with a hydroperoxy-PLP adduct at Cα. Attack of Lys221 at C4’ of PLP at this point would lead to formation of 3 after consumption of one molecule of dioxygen and produce one molecule of H2O2. If water is excluded from the catalytic center and Lys221 does not attack prematurely, loss of the first molecule of peroxide leads to the second quinonoid intermediate (Q2), which reacts with a second molecule of dioxygen. Recombination of superoxide with radical VII after intersystem crossing (ISC) could lead to the diene VIII (Figure 7; it could also result in a hydroperoxy intermediate) and release of a second molecule of H2O2. At this point, Lys221 could react with


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Page 32 of 48

VIII to release the intermediate 4, which is the product of the MppP homolog Ind4 21, from PLP. Intermediate 4 would remain bound in the enzyme active site as noncovalent complex IX. Evidence that hydroxylation takes place on the enzyme rather than by a non-enzymatic hydrolysis lies in the stereoselective nature of the formation of 1. It will be interesting to learn why 4 is released from Ind4, but retained for enzyme-mediated hydration in MppP. Protonation of 4 leads to the imine complex (X), and hydration would lead to the alcohol (XI), perhaps mediated by His29. It should be noted that the hydration step could happen earlier in the mechanism. Finally, the hydroxylated enamine would be released from the enzyme and nonenzymatically deaminated to give 1 and ammonia. Pre-steady state kinetics and deuterium labelling studies are underway to test the current hypothetical mechanism, and if possible, assign rate constants to the observable steps.


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Biochemistry

ASSOCIATED CONTENT Supporting Information. Figure S1 shows the raw data for the catalase experiment described in the text (one set of L-Arg concentrations of the three replicates). Figure S2 shows the mass spectra associated with the isotopic labelling experiment. Figure S3 shows the 1H NMR spectra used to estimate product ratios in reactions of wild-type and mutant forms of MppP with L-Arg. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed: Nicholas R. Silvaggi, Ph.D.: 3210 North Cramer Street, Milwaukee, WI 53211, 414-229-2647, [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by grant CHE-1606842 from the National Science Foundation, Division of Chemistry. ACKNOWLEDGMENT Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was


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supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817).

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[6] Whiting, A. K., Boldt, Y. R., Hendrich, M. P., Wackett, L. P., and Que, L., Jr. (1996) Manganese(II)-dependent

extradiol-cleaving

catechol

dioxygenase

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[20] Han, L., Schwabacher, A. W., Moran, G. R., and Silvaggi, N. R. (2015) Streptomyces wadayamensis MppP Is a Pyridoxal 5'-Phosphate-Dependent

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[28] Meister, A. (1952) Enzymatic preparation of alpha-keto acids, J. Biol. Chem. 197, 309317. [29] Otwinowski, Z., and Minor, W. (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode, Methods Enzymol. 276, 307-326. [30] Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R., and Leslie, A. G. (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM, Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 271-281. [31] McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J Appl Crystallogr 40, 658-674. [32] Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments, Acta Crystallogr D Biol Crystallogr 67, 235-242. [33] Afonine, P. V., Mustyakimov, M., Grosse-Kunstleve, R. W., Moriarty, N. W., Langan, P., and Adams, P. D. (2010) Joint X-ray and neutron refinement with phenix.refine, Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 1153-1163. [34] Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H.


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Table of Contents use only.


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260x76mm (300 x 300 DPI)


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

The L-Arg-bound SwMppP dimer with one protomer shown in cartoon representation, and the other as a solvent-accessible surface (A). The large and small domains are colored blue and green, respectively. The Nand C-termini are labeled. The white arrow and yellow sphere denote the position of Leu23, the first residue visible in the internal aldimine structure (PDB ID 5DJ1 20). Upon substrate binding, the N-terminus orders into a short α-helix (purple) that covers the catalytic center and sequesters it from bulk solvent. A stereoview of the N-terminus shows the electron density for this portion of the structure (B). The yellow sphere indicates the position of Leu23. None of the section in purple is visible in the unliganded or D-Argbound structures of the enzyme. The 2|Fo| - |Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo| - |Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. This figure was prepared using the POVSCRIPT+ modification of MOLSCRIPT41, 42 and POV-Ray (www.povray.org). 177x93mm (300 x 300 DPI)


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Stereoview of the SwMppP active site with the substrate, L-Arg, bound as the external aldimine (A). The 2|Fo| - |Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo| - |Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. The Cα trace and carbon atoms of the main protomer (that containing the catalytic Lys221) are colored light blue, and those of the other protomer in the dimer are colored light green. Residue labels with an asterisk (*) also denote the other protomer. The catalytic Lys221 and the PLP cofactor are shown as sticks with orange carbon atoms. L-Arg is also shown as sticks, but with light purple carbon atoms. Selected solvent molecules are shown as transparent blue spheres. This figure was prepared using the POVSCRIPT+ modification of MOLSCRIPT41, 42 and POV-Ray. The 3-dimensional figures are reduced to schematic form (B) in order to highlight relevant interactions and their distances (Å). Hydrogen bonds and electrostatic interactions are shown as green dotted lines, and distances that do not correspond to any particular noncovalent interaction are shown as grey dotted lines. Panel B and subsequent schematics were created with MarvinSketch (ChemAxon) and Adobe Illustrator CC 2017.


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

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Biochemistry

Stereoview of the internal aldimine form of SwMppP, shown as a grey Cα trace with the internal aldimine (K221-PLP) shown as sticks with yellow carbon atoms, superimposed on the structure of the external aldimine with L-Arg, which is colored as in Figure 2. The chloride ion present in the internal aldimine structure is shown as a green sphere, and water molecules belonging to this structure are shown as transparent yellow spheres. One of these water molecules is displaced by the guanidinium group of L-Arg. 132x66mm (300 x 300 DPI)


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

Stereoview of 1 bound in the active site of SwMppP (A), showing the PLP covalently bound to the enzyme as the internal aldimine. Thr12 and Glu15 interact with the guanidinium and α-carboxylate groups, respectively. His29 interacts with the hydroxyl group of 1. The 2|Fo|-|Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo|-|Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. The protein and solvent are colored as in Figure 2; the product, 1, is shown as sticks with tan carbon atoms. Schematic view of the active site (B) showing potential hydrogen bonding or electrostatic interactions as green, dashed lines. 134x177mm (300 x 300 DPI)


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Biochemistry

Stereoview of the active site in the SwMppP•3 complex (A), showing the PLP covalently bound to the enzyme as the internal aldimine. As in the complex with 1, Thr12 and Glu15 interact with the guanidinium and α-carboxylate groups of 3, respectively. The 2|Fo|-|Fc| electron density map contoured at 1.0σ is shown as magenta mesh, and the 2|Fo|-|Fc| simulated annealing composite omit map, also contoured at 1.0σ, is shown as green mesh. Coloring is as in Figure 2, except that the carbon atoms of 3 are colored yellow. Schematic view of the active site showing potential hydrogen bonding or electrostatic interactions as green, dashed lines (B). 133x183mm (300 x 300 DPI)


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

Orthogonal views of the external aldimine with L-Arg in the structure of SwMppPE15A, colored as in Figure 2. The Ala15 residue is highlighted with a red label. In all respects, save for the absence of four atoms from the glutamate side chain, this structure almost exactly resembles that of the wild-type SwMppP·L-Arg complex. 132x61mm (300 x 300 DPI)


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Revised mechanistic hypothesis consistent with the available experimental observations of SwMppP catalysis. The colored boxes indicate the species that can be detected spectroscopically: I, internal aldimine (415 nm); II, external aldimine (425 nm); Q1, first quinonoid intermediate (510 nm); Q2, second quinonoid intermediate (560 nm). The species in grey boxes are not observed directly. 215x310mm (300 x 300 DPI)


Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not

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