An Oxidative Pathway for Microbial Utilization of Methylphosphonic

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An Oxidative Pathway for Microbial Utilization of Methylphosphonic Acid as a Phosphate Source Simanga Gama, Margret Vogt, Thomas Kalina, Kendall Hupp, Friedrich Hammerschmidt, Katharina Pallitsch, and David L Zechel ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00024 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Chemical Biology

ACS Chemical Biology Article

An Oxidative Pathway for Microbial Utilization of Methylphosphonic Acid as a Phosphate Source

Simanga Gama1, Margret Vogt2, Thomas Kalina2, Kendall Hupp1, Friedrich Hammerschmidt2, Katharina Pallitsch2*, David L. Zechel1*

1.

Department of Chemistry, Queen’s University, Kingston, Ontario, Canada

2.

Institute of Organic Chemistry, University of Vienna, Vienna, Austria

* Corresponding authors: E-mail: [email protected]; [email protected]

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ACS Chemical Biology 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

Methylphosphonic acid is synthesized by marine bacteria and is a prominent component of dissolved organic phosphorus. Consequently, methylphosphonic acid also serves as a source of inorganic phosphate (Pi) for marine bacteria that are starved of this nutrient. Conversion of methylphosphonic acid into Pi is currently only known to occur through the carbon-phosphorus lyase pathway, yielding methane as a by-product. In this work we describe an oxidative pathway for the catabolism of methylphosphonic acid in Gimesia maris DSM8797. G. maris can use methylphosphonic acid as Pi sources despite lacking a phn operon encoding a carbon-phosphorus lyase pathway. Instead, the genome contains a locus encoding homologs of Fe(II) dependent oxygenases HF130PhnY* and HF130PhnZ which were previously shown to convert 2aminoethylphosphonic acid into glycine and Pi. GmPhnY* and GmPhnZ1 were produced in E. coli and purified for characterization in vitro. The substrate specificities of the enzymes were evaluated with a panel of synthetic phosphonates. By 31P-NMR spectroscopy it is demonstrated that the GmPhnY* converts methylphosphonic acid to hydroxymethylphosphonic acid, which in turn is oxidized by GmPhnZ1 to produce formic acid and Pi. In contrast, 2-aminoethylphosphonic acid is not a substrate for GmPhnY* and is therefore not a substrate for this pathway. These results thus reveal a new metabolic fate for methylphosphonic acid.


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Since the 1970s researchers have recorded the occurrence of supersaturating concentrations of methane in shallow, oxygen-rich layers of the world’s oceans 1. This phenomenon became known as the ‘ocean methane paradox’ 2, as the excess methane could not be attributed to pollution from gas extraction, or to microbial methanogenesis, which is limited to anaerobic environments. It is now known that a significant source of ocean and freshwater methane is derived from microbial biosynthesis and catabolism of methylphosphonic acid 1

2-6.

Like all natural organophosphonic

acids (Pn), the biosynthesis of 1 begins with C-P bond formation through the isomerization of phosphoenolpyruvate to phosphonopyruvate by phosphoenolpyruvate mutase and ends with the conversion of 2-hydroxyethylphosphonic acid to 1 by the mononuclear Fe(II) dependent oxygenase MPnS 7,8. Some of the most abundant marine bacterial species, such as those of the Nitrosopumilus and Pelagibacter genera, actively synthesize 1 and esterify this molecule with cell wall polysaccharides 9. Such Pn biomaterial makes a significant contribution to the phosphorus component of marine and freshwater dissolved organic matter (DOM) limiting nutrient in marine and aquatic environments concentrations

13,

12,

2,10,11.

Because Pi is a

at times dropping to picomolar

the ability of microbes to use DOM associated Pn as alternative source of Pi

offers a significant evolutionary advantage 6. Microbial Pn utilization can occur through several catabolic pathways that feature different mechanisms of cleaving the C-P bond to release Pi

14.

Hydrolytic mechanisms, found in

phosphonatase (PhnX), phosphonoacetate hydrolase (PhnY), and phosphonopyruvate hydrolase (PalA) pathways, all require substrates with a beta-carbonyl in order to stabilize a carbanion leaving group upon C-P bond cleavage. For this reason, these pathways are unable to catabolize 1. In contrast, the multienzyme carbon-phosphorus (C-P) lyase pathway is notable for its ability to convert a wide variety of unactivated Pn, including 1, into the corresponding hydrocarbons and Pi 15,16.

The C-P lyase pathway is encoded by the phn operon, which is comprised of 14 genes −3− ACS Paragon Plus Environment


(phnCDEFGHIJKLMNOP) in E. coli

17.

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The phn operon encodes not only discrete enzymatic

reactions, but also a protein complex with the composition Phn(GHIJ)2K18,19. Within the cell, 1 undergoes ribosylation by PhnI (in the presence of PhnG, PhnH, and PhnL) and dephosphorylation by PhnM to produce 5-phospho--D-ribosyl-1-methylphosphonate 2 (Scheme 1A)

20.

2 is the

substrate for homolytic15 C-P bond cleavage by the radical SAM and iron-sulfur cluster (Fe-S) dependent enzyme PhnJ21, yielding methane and -D-ribosyl-1,2-cyclic phosphate 3 ultimately converted to 5-phospho--D-ribosyl pyrophosphate (PRPP)

22,23,

20.

3 is

a universal ribosyl

donor that is used to synthesize nucleotides, histidine, and tryptophan. The C-P lyase pathway is commonly found in marine phytoplankton and is actively expressed due to the low Pi levels that persist in marine environments 4,6,24,25. It is estimated that turnover of only 0.25% of the available amounts of 1 appended to DOM by marine C-P lyase pathways can account for the ocean methane paradox 2. Until now C-P lyase was the only microbial pathway that was known to enable the use of 1 as a Pi source

26.

However, a recently discovered oxidative pathway has, in principle, the

reactivity to cleave the C-P bond of 1. Through functional screening of a cosmid library constructed from marine metagenomic DNA, Martinez and co-workers isolated a cosmid (HF130_AEPn_1) containing a pair of genes, phnY* and phnZ, that encoded the utilization of 4 as a Pi source (Scheme 1B)

25.

HF130PhnY* is a non-heme iron / -ketoglutarate dependent dioxygenase that

hydroxylates the -carbon of 4 to form (R)-5

27,28.

HF130PhnZ, an outlying member of the HD

superfamily of phosphohydrolases, subsequently uses a mixed valence (FeII/FeIII) di-iron cofactor 29

to oxidatively cleave the C-P bond of (R)-5, forming glycine and Pi as products (Scheme 1B)

27,28.

Although mixed valence diiron oxygenases are predicted to form a large and diverse subgroup


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within the HD superfamily 29, only one other member of this family, the mammalian enzyme myoinositol monooxygenase, has been functionally characterized 30. The PhnY* / PhnZ sequences occur frequently in marine bacteria, rivalling the frequency of genes encoding C-P lyase

25,31,32.

In 2010 Martinez and co-workers identified genes encoding

one PhnY* and two PhnZ homologs in the planktomycete Gimesia maris DSM8797 (formerly known as Planctomyces maris) GmPhnZ1

(WP_002646829.1)

25.

The genes encoding GmPhnY* (WP_002646828.1) and

are

encoded

by

adjacent

genes,

while

GmPhnZ2

(WP_002648691.1) is encoded at a different locus that features transaminase PhnW and phosphonatase PhnX genes encoding the degradation of 4. As expected, G. maris DSM8797 was able to grow on 4 as a Pi source. However, G. maris DSM8797 was also able to use 1 as a Pi source; this was a remarkable finding as this bacterium lacks a phn operon encoding C-P lyase. In this study, we resolve this paradox by demonstrating that 1 utilization by G. maris DSM8797 is enabled by an oxidative pathway comprised of GmPhnY* and GmPhnZ1.

RESULTS AND DISCUSSION GmPhnY* Is Specific for Hydroxylation of Methylphosphonic Acid. The functions of GmPhnY* and GmPhnZ1 were studied in vitro. Codon optimized genes encoding GmPhnY* and GmPhnZ1 bearing C-terminal hexa-histidine tags were successfully expressed in E. coli. The enzymes were purified by Ni-NTA affinity chromatography followed by size exclusion chromatography (Figure S1). To test the activity of GmPhnY*, the enzyme (10 μM) was incubated with 1, -ketoglutarate, and Fe(II) for 16 h at 30 °C (pH 7). The 31P NMR spectrum of 1 exhibits a signal at  = 24.2 ppm relative to phosphoric acid ( = 0.0 ppm) (Figure 1A). The reaction with GmPhnY* produces a new signal at  = 17.8 ppm (Figure 1B). This signal corresponds to



hydroxymethylphosphonic acid 6, the identity of which was confirmed by the addition of synthetic 6 to the NMR sample and observing a corresponding increase in the signal (Figure 1C). The kinetic parameters of GmPhnY* for conversion of  to 6 were determined using GmPhnZ1 as a coupling enzyme to produce Pi (Figure S2). Saturating behaviour with respect to Fe(II) is observed, with an apparent KM of 4  1 M, suggesting that the enzyme is not fully metal ion loaded after purification (Figure S3A). In the presence of saturating Fe(II) (100 M) Michaelis-Menten rate behaviour is observed when varying the concentration of 1 (Figure S3C). With -ketoglutarate held constant at 2 mM, apparent kinetic parameters of kcat = 0.36 ± 0.1 s-1, KM = 1.5 ± 0.1 mM, and kcat/KM = (2.4 ± 0.2) × 102 M-1 s-1 are observed for 1 (pH = 7, 25 °C). Similarly, in the presence of saturating 1, the apparent kinetic parameters for -ketoglutarate are kcat = 0.42 s-1, KM = 11 ± 3 M, and kcat/KM = (8 ± 2) × 104 M-1 s-1 (Figure S3B). An additional nine other Pn were tested as substrates with GmPhnY*, including ethylphosphonic acid (7) and aminoalkylphosphonic acids such as 4, aminomethylphosphonic acid (8), phosphonoalanine (13), and glyphosate (14) (Table S1). However, according to

31P

NMR spectroscopy, none of these Pn compounds were converted to

new products by GmPhnY*. These results indicate that GmPhnY* is specific for hydroxylation of 1. It is notable that 6 has been observed previously as an intermediate in the biosynthesis of phosphinothricin

33.

In this case 6 is formed by oxidative C-C bond cleavage of 2-

hydroxyethylphosphonic acid by hydroxyethylphosphonic acid dioxygenase (HEPD) 34, a homolog of MPnS. Interestingly, 6 has also been observed by 31P NMR spectroscopy as a major constituent of DOM along with 1 and 2-hydroxyethylphosphonic acid 2. GmPhnZ1 Oxidatively Converts Hydroxymethylphosphonic Acid Into Formate and Pi. The reaction of GmPhnZ1 was initially characterized by 31P NMR spectroscopy. GmPhnZ1 was incubated with 6 in the presence of Fe(II) and ascorbate at 30 °C (pH 7). The resulting 31P NMR


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spectrum of the reaction mixture revealed that the  = 17.8 ppm signal corresponding to 6 had been converted to a signal at  = 2.9 ppm corresponding to Pi (Figure 1D). The corresponding 1H NMR spectrum of the reaction exhibited a downfield signal at  = 8.3 ppm, consistent with aldehydic proton of formate (Figure 2B). This was confirmed by adding authentic formate to the sample and observing an increase in the  = 8.3 ppm signal (Figure 2C). Therefore, the overall oxidation of 1 by GmPhnY* and GmPhnZ1 produces Pi and formate as products. The rate of reaction of GmPhnZ1 with 6 was measured in a stopped assay format using Malachite Green to detect the Pi formed. The GmPhnZ1 reaction rate exhibited saturating behaviour with respect to the concentration of Fe(II), with an apparent KM value of 10 ± 2 µM, implying that the purified enzyme is not fully loaded with two Fe ions per active site (Figure S4A). In the presence of saturating Fe(II) (pH = 7, 25 °C), the GmPhnZ1 reaction rate with 6 exhibited Michaelis-Menten kinetic behaviour with the kinetic parameters kcat = 0.55 ± 0.01 s-1, KM = 26 ± 3 µM, and kcat/KM = (2.1 ± 0.03) × 104 M-1 s-1 (Figure S4B). GmPhnZ1 was tested for reactivity with nine additional Pn (Table S2). Surprisingly, GmPhnZ1 was also reactive towards (R)-5, the substrate for HF130PhnZ, converting this compound to Pi as shown by 31P NMR spectroscopy (Figure S5). The kinetic parameters with (R)-5 are kcat = 2.5 ± 0.01 s-1, KM = 130 ± 16 M, and kcat/KM = (2.0 ± 0.2) × 104 M-1 s-1 (Figure S6), indicating that the efficiency with this substrate is equivalent to 6. In contrast, GmPhnZ1 was unreactive towards (S)-5 (data not shown), indicating that GmPhnZ1 shares the same stereospecificity for C-H bond cleavage as HF130PhnZ 28. Additionally, GmPhnZ1 was reactive towards (R)-1-hydroxyethylphosphonic acid (R)-17 (Figure S7), yielding kinetic parameters of kcat = 1.54 ± 0.03 s-1, KM = 280 ± 17 M, and kcat/KM = (1.8 ± 0.1) ×104 M-1 s-1 (Figure S8). This is notable because HF130PhnZ is unreactive towards (R)-17

28

as well as 6 (data not shown). This



indicates that unlike GmPhnZ1, the HF130PhnZ homolog has a high requirement for a substrate amino group. PhnY* Sequence Similarity Network. The determination of substrate specificities for GmPhnY* and GmPhnZ1 provides an opportunity to examine the relationship between sequence and function. GmPhnY* is 36% identical to HF130PhnY* and shares the predicted Fe(II) and ketoglutarate binding residues that are conserved in non-heme iron / KG family of dioxygenases (Figure S9). To further examine the relatedness of PhnY* dioxygenases, a sequence similarity network was generated using the EFI-EST server 35. A BLAST analysis of HF130PhnY* provided 1000 sequences that could be resolved into a network of 49 clusters where each cluster contains enzymes with greater than 40% sequence identity (Figure 3A). Only 3 clusters contain sequences with experimentally assigned functions. This includes the main cluster (Figure 3A, red) which divides into three connected sub-clusters. Sub-cluster (a) contains the HF130PhnY sequence, while the GmPhnY* sequence is found in sub-cluster (b). Sub-cluster (b) also contains the PhnY* sequences from Prochloroccus marinus strains MIT9301 and MIT9303. The genes encoding Pm9301PhnY* (WP_011863204.1) and Pm9303PhnY* (ABM77875.1) appear in loci that contain phnZ sequences as well as ptxABCD cistrons that encode utilization of phosphite as a Pi source 25,32.

Interestingly, sub-cluster (c) is distinguished by PhnY* sequences (e.g.: OUT50259.1,

ARV58119.1) that are encoded by loci that feature genes for phosphoenolpyruvate mutase, phosphonopyruvate decarboxylase, and a pyridoxal phosphate dependent transaminase. This combination, previously noted by Metcalf and coworkers based on an analysis of phosphoenolpyruvate mutase sequences 36, is predicted to encode the biosynthesis of 5, which is a known component of phosphonolipids 37,38. Other clusters containing enzymes of known function include (d), containing two dioxygenases (LolE-1 and LolE-2; UniProt IDs Q5MNH9 and


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Q5MNH2 respectively) involved in the biosynthesis of the fungal alkaloid loline

39,

and (e),

containing HtxA (AAC71711.1), the dioxygenase that oxidizes hypophosphite to phosphite, thereby enabling bacteria to use this compound as a Pi source 40,41. Surprisingly, other KG / nonheme Fe(II) dioxygenases that are known to perform -hydroxylation of phosphonates in biosynthetic contexts, such as the fosfazinomycin enzyme FzmG (WP_053691445.1) dehydrophos

enzymes

DhpA

(ACZ13452.1)

and

DhpJ

(ACZ13461.1)

43;

42;

and

the the

hydroxyphosphonocystoximic acid enzyme HpxV (WP_030990672.1) 44, do not appear within this PhnY* network. This indicates that distantly related KG / non-heme Fe(II) dependent dioxygenases can catalyze -hydroxylation of phosphonates, and implies that this chemistry has evolved independently within this superfamily. PhnZ Sequence Similarity Network. Because structures of HF130PhnZ bound to (R)-5 are available, differences in substrate specificity with GmPhnZ1 can be rationalized in greater detail. GmPhnZ1 is 36% identical to HF130PhnZ (Figure S10) and shares all of the conserved active site residues, with the notable exception of the flexible active site loop near the N-terminus (residues 23 to 28). In HF130PhnZ this loop contains the residues Y24 and E27 which mediate an induced fit mechanism for binding (R)-5 28. In an unreactive HF130PhnZ conformation, Y24 forms a ligand interaction with one Fe ion, thereby blocking O2 binding and reduction to superoxide. Upon binding (R)-5 at the adjacent Fe ion, interaction of E27 with the 2-amino group of the substrate causes Y24 to be flipped out of the active site, thereby exposing the O2 binding site for catalysis. While E27 is conserved in GmPhnZ1, the remainder of the loop sequence is not conserved and appears to be one amino acid shorter. This variation in loop sequence may contribute to the differing reactivities of HF130PhnZ and GmPhnZ1 towards 6. The relatedness of PhnZ enzymes was further examined through a sequence similarity network based on 5000 homologs of



HF130PhnZ (Figure 3B). The PhnZ sequences could be resolved into 9 clusters where each cluster contains enzymes with greater than 45% sequence identity. Analogous to what was observed with PhnY*, the PhnZ network is dominated by a large cluster (shown in red) that divides into two connected sub-clusters that contain the GmPhnZ1 (a) and HF130PhnZ (b) sequences. Sub-cluster (a) also contains the P. marinus enzymes Pm9301PhnZ (WP_011863205.1) and Pm9303PhnZ (ABM77874.1). Although sub-clusters (a) and (b) generally contain PhnZ sequences that are encoded in genomes alongside PhnY*, this is not always the case, as shown by the appearance of the phosphonatase pathway associated GmPhnZ2 in sub-cluster (a). None of the other clusters have assigned functions (based on SwissProt annotations). However, sub-cluster (c) is notable for containing PhnZ genes (e.g.: ORW68566.1, WP_069862562.1) that are associated genes encoding components of C-P lyase.

CONCLUSIONS In summary, a new microbial oxidative pathway for utilization of 1 as a source of Pi has been established. While catabolism of 1 by microbial C-P lyase pathways results in the loss of carbon as methane, the oxidation of 1 into formate by GmPhnY* and GmPhnZ raises the possibility that 1 could also be used as a source of carbon or reducing equivalents 45-47. Sequence similarity network analyses of PhnY* and PhnZ reveal numerous clusters with unknown functions, but in both cases a dominant cluster is found that corresponds to oxidative Pn catabolism or modification. The size of these dominant clusters implies the existence of new substrate specificities that would allow PhnY*/PhnZ to process other simple Pn as Pi sources, and that the versatility of this pathway has yet to be fully appreciated.

METHODS − 10 − ACS Paragon Plus Environment

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General. All reagents were purchased from Sigma-Aldrich Canada or BioShop Canada, Inc., unless otherwise specified. The Malachite green reagent was prepared as described previously 48. 31P

NMR spectra were recorded on a Bruker Avance-300 spectrometer and referenced to H3PO4

( = 0 ppm). Ni-NTA resin was obtained from Qiagen (Canada) and Sephacryl S-200 resin was obtained from GE Healthcare Life Sciences (USA). Synthetic genes encoding GmPhnY* and GmPhnZ were obtained from ATUM (Newark, California). The extinction coefficients for GmPhnY* and GmPhnZ were calculated from their respective amino acid sequences using the ProtParam tool from ExPASy (https://web.expasy.org/protparam/) 49. The synthesis and analytical characterization of organophosphonic acid substrates for GmPhnY* and GmPhnZ1 is described in the Supporting Information. Production of GmPhnY* and GmPhnZ1. The genes encoding G. maris DSM8797 PhnY* (GenBank accession: WP_002646828) and PhnZ1 (WP_002646829) were synthesized with codons optimized for expression in E. coli. Each gene encoded an additional GSGSGSHHHHHH sequence at the C-terminus of each protein. The genes were cloned into the pD451 plasmid yielding pD451-SR:319166 encoding GmPhnY* and pD451-SR:319168 encoding GmPhnZ1. Each plasmid contains a T7 promoter and a kanamycin resistance gene. For protein expression, the plasmids were transformed into E. coli BL21(DE3) cells and grown overnight at 37 °C on LB-agar supplemented with 50 µg/mL kanamycin. A single colony was used to inoculate 50 mL of LB medium with 50 µg/mL kanamycin, which was then incubated in an air shaker overnight at 37 °C and 200 rpm. From this pre-culture 10 mL was transferred to a 1 L of LB containing 50 µg/mL kanamycin, which was incubated at 37 °C and 200 rpm until the culture reached an OD600 value of 0.6. Isopropyl βD-1-thiogalactopyranoside

(IPTG) was added to a final concentration of 0.5 mM, followed by

incubation of the culture at 30 °C, 200 rpm, for 4 h. The cells were harvested by centrifugation at

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5000  g for 15 min. The cell pellet was flash-frozen in liquid nitrogen and stored at -30 °C until purification. The thawed cell pellet was suspended in buffer A (20 mM Tris-Cl, pH 7.5, 300 mM NaCl, 10 mM imidazole), lysed at 15,000 psi with an EmulsiFlex-C5 cell homogenizer (Avestin, Inc. Canada), then centrifuged at 28,000  g for 30 min at 4 °C. The clarified cell lysate was applied onto a 5 mL Ni-NTA Sepharose column pre-equilibrated with buffer A. The column was connected to an AKTA FPLC system then washed with 10 column volumes of buffer A at 5 mL / min, followed by a linear gradient to 100% buffer B over 10 column volumes (20 mM Tris-Cl, pH 7.5, 300 mM NaCl, 500 mM imidazole). Pure protein fractions were subsequently combined, concentrated by ultrafiltration, and purified by size exclusion chromatography (Sephacryl S-200 HR, AKTA XK-16 column, 60 cm). Fractions containing purified proteins were concentrated by ultrafiltration then dialyzed into 25 mM Tris-Cl pH 7.5, 150 mM KCl, 10% (v/v) glycerol. Enzyme concentrations were determined by absorbance at 280 nm using the extinction coefficients

ε280

= 47,120 M-1 cm-1 for GmPhnY* and ε280 = 18,575 M-1 cm-1 for GmPhnZ1. 31P

NMR spectroscopic analysis of enzyme reactions. The GmPhnY* reaction contained 2 mM

-ketoglutarate, 1 mM substrate, 200 μM TCEP, 50 μM ascorbic acid, 100 μM ammonium iron(II) sulfate, 1 mg/ml bovine serum albumin, and 10 μM GmPhnY* in 50 mM MOPS-HCl, 150 mM NaCl, 10% (v/v) glycerol, pH 7 (total volume = 600 L). The reaction was incubated at 30 °C for 16 h, whereupon D2O [10% (v/v) final] and K2HPO3 (1 mM final, as an internal reference) were added.

31P

NMR spectra were recorded on a 300 MHz Bruker Avance-300 spectrometer (1024

scans, 121 MHz, spectral window of -50 to 250 ppm). The reaction mixture for GmPhnZ1 was identical with the exception that -ketoglutarate was omitted. Additionally, K2HPO3 was omitted from the GmPhnZ1 NMR sample.


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Kinetic analysis of GmPhnY* and GmPhnZ1. The GmPhnY* rate of conversion of 1 to 6 was monitored by coupling the reaction to the GmPhnZ1 reaction. The Pi ultimately produced in this coupled reaction was quantified using Malachite green 48. The reaction mixture contained 1, 2 mM -ketoglutarate, 100 μM TCEP, 200 μM ascorbic acid, 100 μM ammonium iron(II) sulfate, 1 mg/mL bovine serum albumin, 50 mM MOPS-HCl, 150 mM NaCl, 10% (v/v) glycerol, pH 7 (total volume = 350 μL). The concentration of 1 was varied from 1/5 to 5  the final KM value. The reaction mixture, except for ammonium iron(II) sulfate, was preincubated at 25ºC for 2 min, whereupon the reaction was initiated by the addition of iron followed by a mixture of GmPhnY* and GmPhnZ1 to final concentrations of 1 μM and 10 μM, respectively. The concentration of GmPhnZ1 was sufficient to ensure a linear response in reaction rate with respect to GmPhnY* concentration (Figure S2). To measure initial rates, the production of Pi was monitored over 2 min by removing 50 L aliquots at 30 s intervals. The reaction aliquots were combined with 50 L Malachite green reagent. The Malachite green solution contained 0.063% (w/v) Malachite green, 0.1% (v/v) Tween 20, and 2.1% (w/v) ammonium molybdate in 2 M HCl. The assay is nonlinear at very low Pi concentrations, presumably due to incomplete Pi complexation by the Malachite green/ammonium molybdate reagent

50,

therefore 5 μM K2HPO4 was added to the solution to

ensure that incremental Pi produced by GmPhnZ1 was in the linear range. The absorbance was measured at 630 nm in a plate reader (Molecular Devices) after a 10 min incubation at room temperature. Absorbance values were converted to Pi concentrations using a standard curve, and initial rates (Vo) determined by a linear fit to the plot of [Pi] vs time. The apparent kinetic parameters for 1 at a fixed concentration of -ketoglutarate (2 mM) were determined by a fit of the plot of Vo/[Eo] vs [1] to the Michaelis-Menten equation, where [Eo] is the total enzyme concentration.



The GmPhnZ1 catalyzed conversion of 6 to Pi was also monitored using the Malachite green assay. The reaction mixture contained 6, 100 μM TCEP, 3 μM ascorbic acid, 100 μM ammonium iron(II) sulfate, 1 mg/mL BSA in 50 mM MOPS-HCl, 150 mM NaCl, 10% (v/v) glycerol, pH 7 (total volume = 300 μL). The concentration of 6 ranged from 5 μM to 2000 μM. This reaction mixture, except for ammonium iron(II) sulfate, was preincubated at 25 °C for 2 min, whereupon the reaction was initiated by the addition of ammonium iron(II) sulfate followed by GmPhnZ (to a final concentration of 667 nM). The production of Pi was monitored over 2 min by removing 50 L aliquots at 30 s intervals. The reaction aliquots were assayed for Pi using Malachite Green as described above. The kinetic analysis of GmPhnZ1 with (R)-5 and (R)-17 was performed in a likewise fashion. The kinetic parameters were determined using the Michaelis-Menten equation.

ACCESSION CODES GmPhnY*: WP_002646828 GmPhnZ1: WP_002646829

ASSOCIATED CONTENT The following Supporting Information is available free of charge via the internet. Supporting Information: Substrates tested with GmPhnY* and GmPhnZ1 (Tables S1 and S2), SDS-PAGE of purified GmPhnY* and GmPhnZ1 (Figure S1), response of GmPhnY* reaction rate using GmPhnZ as a coupling enzyme (Figure S2); kinetic analysis of the GmPhnY* reaction with 1 (Figure S3); kinetic analysis of the GmPhnZ1 reaction with 6, (R)-5, and (R)-17 (Figures S4 to S6); sequence alignment of HF130PhnY* and GmPhnY* (Figure S7); sequence alignment of HF130PhnZ and


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GmPhnZ1 (Figure S8); synthesis and analytical characterization of substrates; NMR spectra of synthesized compounds and intermediates.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected]; [email protected]

ORCID Simanga Gama: 0000-0002-0809-7084 Friedrich Hammerschmidt: 0000-0003-2193-1405 Katharina Pallitsch: 0000-0003-2648-1044 David L. Zechel: 0000-0002-1570-599X ACKNOWLEDGMENTS D.L. Zechel thanks the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support (492945-2016 and 03695-2016). K. Pallitsch thanks the Austrian Science Fund (FWF) for support (P27987-N28).



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FIGURES Figure 1

Figure 1.

31P-NMR

spectroscopic analysis of GmPhnY* and GmPhnZ1 reactions (121 MHz,

referenced to H3PO4,  = 0 ppm). (A) Spectrum for methylphosphonic acid (1). The signal for 1 appears at  = 24.2 ppm. ( Reaction of GmPhnY* with 1. The signal for hydroxymethylphosphonic acid (6) appears at  = 17.8 ppm. (C) Addition of synthetic 6 to the GmPhnY* reaction with 1. (D) Reaction of GmPhnZ1 with 6. In spectra A, B, and C, 1 mM K2HPO3 is included as an internal reference ( = 2.9 ppm). In spectrum D, the Pi signal is derived solely from the GmPhnZ1 reaction.



Figure 2

Figure 2. (A) 1H-NMR spectrum of the GmPhnZ1 reaction prior to adding enzyme. (B) Spectrum of the GmPhnZ1 reaction with 6. The signal for formate (appearing at  = 8.3 ppm) is marked with an asterisk (*). (C) Spectrum after the addition of formate.


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Figure 3

Figure 3. (A) Sequence similarity network for PhnY* homologs. The network is based on 1000 unique sequences retrieved from a BLAST analysis of HF130PhnY*. Individual clusters, each with a unique color, correspond to sequences with >40% identity (alignment score = 60). (B) Sequence similarity network for PhnZ homologs. The network is based on 5000 unique sequences retrieved from a BLAST analysis of HF130PhnZ. Individual clusters correspond to sequences with >45% identity (alignment score = 40). Clusters that are discussed in the text are circled and labelled. The networks were created using the EFI-EST server 35 and visualized in Cytoscape (v 3.6.1).



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SCHEMES O HO P O OH

A C-P lyase

O H3C P OH OH

KG, O2

1

O

O O P CH3 OH OH OH 2

succinate, CO2

GmPhnY* (FeII)

HO

SAM

CH4

3

H

O2

O P OH OH

H2 N

4

HF130PhnY* (FeII) H2 N

KG, succinate, CO2 O2

HO

O P OH OH

OH O

6

B

O OH O

5'-dAdo,

GmPhnZ1 (FeII/FeIII)

O P OH OH

O HO P O OH

PhnJ [4Fe-4S]

HF130PhnZ (FeII / FeIII) O2

OH

H2 N

O

+

HO

O P O

O HO P OH OH

O + HO P OH OH

(R)-5

Scheme 1. (A) Catabolism of methylphosphonic acid 1 by the C-P lyase and GmPhnY* / PhnZ1 pathways. (B) Catabolism of 2-aminoethylphosphonic acid 4 by the HF130PhnY* / PhnZ pathway.


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