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Functional and Structural Analysis of Phenazine OMethyltransferase LaPhzM from Lysobacter antibioticus OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin Jiasong Jiang, Daisy Guiza Beltran, Andrew Schacht, Stephen Wright, Limei Zhang, and Liangcheng Du ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00062 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ACS Chemical Biology

1

Functional and Structural Analysis of Phenazine O-Methyltransferase LaPhzM from

2

Lysobacter antibioticus OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin

3 4

Jiasong Jiang,† Daisy Guiza Beltran,‡ Andrew Schacht,‡ Stephen Wright,† Limei

5

Zhang,*,‡,§ and Liangcheng Du*,†

6 7

†

8

‡

9

Integrated Biomolecular Communication, University of Nebraska-Lincoln, NE 68588, USA

Departments of Chemistry, University of Nebraska-Lincoln, NE 68588, USA Departments of Biochemistry,

§

Redox Biology Center and the Nebraska Center for

10 11

Note: JJ and DGB contributed equally.

12 13 14 15 16

1 ACS Paragon Plus Environment

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

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ABSTRACT: Myxin is a well-known antibiotic that had been used for decades. It

18

belongs to the phenazine natural products that exhibit various biological activities, which are

19

often dictated by the decorating groups on the heteroaromatic three-ring system. The three

20

rings of myxin carry a number of decorations, including an unusual aromatic N5,N10-dioxide.

21

We previously showed that phenazine 1,6-dicarboxylic acid (PDC) is the direct precursor of

22

myxin and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative

23

hydroxylation

24

(1.6-dihydroxy-N5,N10-dioxide phenazine). In this work, we identified LaPhzM gene from

25

Lysobacter antibioticus OH13 and demonstrated that LaPhzM encodes a SAM-dependent

26

O-methyltransferase converting iodinin to myxin. The results further showed that LaPhzM is

27

responsible for both mono-methoxy and di-methoxy formation in all phenazine compounds

28

isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing

29

O-methylation of phenazines with non-, mono-, or di-N-oxide. In addition, we demonstrated a

30

one-pot biosynthesis of myxin by in vitro reconstitution of the three phenazine-ring

31

decorating enzymes. Finally, we determined the X-ray crystal structure of LaPhzM with a

32

bound cofactor at 1.4 Å resolution. The structure provided molecular insights into the activity

33

and selectivity of the first characterized phenazine O-methyltransferase. These results will

34

facilitate future exploitation of the thousands phenazines as new antibiotics through

35

metabolic engineering and chemoenzymatic syntheses.

and

aromatic

N-oxidations

of

PDC


to

produce

iodinin

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Phenazines are a large group of heterotricyclic N-containing aromatic compounds. Over

37

6000 phenazines have been isolated from living organisms or chemically synthesized during

38

the last one and half century.1-4 Among them, several hundreds were reported to possess

39

biological activities, including antimicrobial, antimalarial, and anticancer functions. The first

40

phenazine, pyocyanin (1-hydroxy-N5-methylphenazine), was isolated from the Gram

41

negative opportunistic pathogen Pseudomonas aeruginosa in the late 19th century (Figure

42

1).1 This bacterial pathogen uses pyocyanin as a virulence factor for infection,5-6 and recent

43

work has showed that pyocyanin is a redox-active metabolite facilitating extracellular

44

electron transfer and survival in anoxic environments, which is critical for biofilm

45

development.7-11

46

All phenazines share a three-ring core (dibenzopyrazine) but differ at the decorations on

47

the ring system. The decorating groups, including carboxylate, hydroxy, amide, N-methyl,

48

O-methyl (methyoxy), and prenyl groups, dictate the difference in phenazine biological

49

activities.12 Recent studies also showed that phenazines of P. aeruginosa could be selectively

50

modified by surrounding organisms as a means to control the pathogen’s biofilms.13 The

51

biosynthetic mechanism for the phenazine core has been extensively investigated by several

52

groups.1-4,

53

1-carboxylic acid (PCA) and/or phenazine-1,6-dicarboxylic acid (PDC) (Figure 1). In

54

contrast, relatively little has been done on the molecular mechanism underlying the

55

decorations on the rings. Phenazine natural products with a hydroxy group at the six

56

modifiable carbons of the phenazine core have been isolated from Pseudomonas species.1

57

Interestingly, the hydroxy groups are rarely methylated, although several well-known

14-15

Five to seven genes are required to convert chorismic acid to phenazine



58

Pseudomonas products contain an N-methyl group, such as pyocyanin and aeruginosins

59

(Figure 1). In L. antibioticus OH13, we previously isolated six phenazines (1-6) including the

60

well-known antibiotic myxin (1-hydroxy-6-methoxyphenazine- N5,N10-dioxide).16 Myxin

61

possesses potent antimicrobial activity with a unique mode of action and was manufactured

62

as a copper (II) complex, Cuprimyxin, which had been used for decades as a topical broad

63

spectrum veterinary antibiotic and antifungal drug.12 Five of the six compounds isolated from

64

L. antibioticus contain one or two O-methyl groups, but no N-methyl group (Figure 1).16 The

65

O-methylation is important for the antibiotic activity of phenazines. For example, myxin and

66

iodinin (1,6-dihydroxyphenazine N5,N10-dioxide) have an identical structure, except that

67

none of the hydroxy groups is methylated in iodinin (Figure 1), but myxin has much higher

68

antimicrobial activity than iodinin in all the tests we conducted.16 While O-methylation is

69

very common in natural product biosynthesis, the O-methylation in myxin and other

70

phenazines had not been investigated.

71

In pyocyanin, two enzymes are involved in the decoration of the phenazine ring during

72

biosynthesis in P. aeruginosa.17-20 The first enzyme (PhzM) is an S-adenosylmethionine

73

(SAM) dependent N-methyltransferase, converting PCA to N5-methyl-PCA. Curiously, PhzM

74

was shown to be active only in the physical presence of PhzS, a flavin-dependent

75

hydroxylase catalyzing a decaroxylative hydroxylation in pyocyanin biosynthesis. The

76

N-methylation product, N5-methyl-PCA, was not isolated in the PhzM reaction. It was

77

proposed that N5-methyl-PCA could be unstable and served as an enzyme-bound

78

intermediate that was further processed by the second enzyme, PhzS, to produce the final

79

product, pyocyanin. The result suggested that a protein-protein interaction between PhzM and 4 ACS Paragon Plus Environment

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PhzS might be required in pyocyanin production, as this interaction could ensure an efficient

81

production of pyocyanin by preventing the formation and release of the unstable intermediate

82

N5-methyl-PCA. The crystal structure of both PhzM and PhzS had been reported.17-20 The 1.8

83

Å dimeric structure of PhzM showed three domains per monomer characteristic of many

84

small molecule methyltransferase but did not contain any ligand. The 2.4 Å crystal structure

85

of PhzS showed that the enzyme belongs to the family of aromatic hydroxylases.

86

Phenazine ring decorations are usually specific to each producing organism. In strain

87

OH13, the LaPhz gene cluster contains four genes that were predicted to be responsible for

88

phenazine ring decoration.16 One gene codes for the FAD-dependent enzyme LaPhzS that

89

catalyzes twice of decarboxylative hydroxylation of PDC, the common precursor of all

90

phenazines in strain OH13 (Figure 1). This reaction converts PDC to 1,6-dihydroxyphenazine

91

(7). The next gene encodes LaPhzNO1, a Baeyer-Villiger-like flavin enzyme that catalyzes

92

N-oxidation at both N5 and N10 of the phenazine core. This enzyme eventually converts

93

1,6-dihydroxyphenazine (7) to 1,6-dihydroxyphenazine N5,N10-dioxide (iodinin, 3). Within

94

the LaPhz gene cluster, there remain two genes (LaPhzM and LaPhzX) that have not been

95

investigated for their function. LaPhzM shows a sequence similarity to genes coding for

96

methyltransferases including pyocyanin PhzM,18 and LaPhzX is similar to genes encoding

97

small monooxygenases that catalyze oxidation of phenolic compounds to corresponding

98

quinones and do not require any metal ion or other cofactors.21-22 Interestingly, although

99

LaPhzM is homologous to pyocyanin PhzM (Figure S1), no N-methyl containing phenazines

100

have been identified from strain OH13. In this work, we showed that LaPhzM is responsible

101

for O-methylation in the biosynthesis of myxin and other phenazine natural products isolated 5 ACS Paragon Plus Environment


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102

from strain OH13. We characterized the SAM-dependent O-methyltransferase LaPhzM

103

through heterologous expression, in vitro assay of the enzyme activity and selectivity, one-pot

104

reconstitution of myxin biosynthesis in vitro, and determination of the crystal structure of

105

LaPhzM complexed with cofactor.

106 107

■ RESULTS AND DISCUSSION

108 109

LaPhzM encodes an O-methyltransferase for production of both mono-methylated

110

and di-methylated phenazines in L. antibioticus OH13. The LaPhzM gene within the

111

phenazine biosynthetic gene cluster LaPhz in strain OH13 was heterologously expressed in E.

112

coli as a 36.7 kDa protein (Figure S2). To study the role of LaPhzM in the biosynthesis of

113

myxin and other phenazines, we incubated LaPhzM with 6-hydroxy-1-methoxyphenazine

114

N5-oxide (2), the main phenazine compound produced by strain OH13.16 The substrate eluted

115

at the retention time of 14.3 min on HPLC, and the reaction yielded one new product with the

116

retention time of 6.8 min (Figure 2 and Figure S3). The production of this product was

117

dependent on LaPhzM and SAM, as neither the boiled LaPhzM nor the lack of SAM

118

produced this compound. The new product had the same retention time as

119

1,6-dimethoxyphenazine N5-oxide (4), which had previously been isolated from strain

120

OH13.16 Mass spectrometry analysis of the new product gave a m/z of 257.07 (Figure S4A),

121

which is also consistent with [M + H]+ for 1,6-dimethoxyphenazine N5-oxide (calculated

122

mass

123

O-methyltransferase for phenazines. Kinetics study of the enzyme gave a KM of 33.6 µM and

256.08).

The

result

demonstrates

that

LaPhzM


is

a

SAM-dependent

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kcat of 0.6 min-1, with Vmax of 2.9 µM min-1, when 6-hydroxy-1-methoxyphenazine N5-oxide

125

(2) was the substrate to produce 1,6-dimethoxyphenazine N5-oxide (4) (Figure S3).

126

In addition to compounds 2 and 4, strain OH13 produces three other O-methyl

127

containing

phenazines,

including

1-hydroxy-6-methoxyphenazine

(6),

128

1,6-dimethoxyphenazine (5), and myxin (1).16 To further understand the role and substrate

129

selectivity of LaPhzM, we tested the enzyme’s activity with dihydroxyl phenazine as

130

substrate (Figure 3).

131

and a m/z of 213.40 for [M + H]+ (Figure 3 and Figure S4B), which is also consistent with the

132

calculated mass of the compound (calculated mass 212.06). After 15 min of reaction, a major

133

peak at 9.8 min appeared. This product co-migrated with 1-hydroxy-6-methoxyphenazin (6)

134

and gave a m/z of 227.02 for [M + H]+ (Figure 3 and Figure S4C), which is consistent with

135

the calculated mass of 6 (calculated mass 226.07). As the reaction continued, the peaks for

136

the substrate (7) and the first product (6) decreased and a third peak at 9.5 min became the

137

predominate product in reaction mixture (Figure 3). This peak had the same retention time as

138

1,6-dimethoxyphenazine (5) and gave a m/z of 241.02 for [M + H]+ (Figure S4D), which is

139

consistent with the calculated mass of 5 (calculated mass 240.09). The results show that

140

LaPhzM is responsible for O-methylation of both the mono-methylated and di-methylated

141

phenazines produced by strain OH13.16

1,6-Dihydroxyphenazine (DHP, 7) gave the retention time of 10.1 min

142

Additionally, we compared the relative reaction rate of LaPhzM toward different

143

substrates (Figure S5). The enzyme exhibited the highest relative activity when the

144

dihydroxy-containing

145

monohydroxy-containing phenazine (10), monohydroxy-monomethoxy-containing phenazine

phenazine

DHP

(7)

was

the


substrate,

followed

by


146

(2), and non-phenazine substrates (8-hydroxyquinoline and 6-hydroxyquinoline).

147

Reconstitution of myxin biosynthesis in vitro. Myxin is perhaps the most interesting

148

phenazine natural product due to its potent antibiotic activity and rich decorations on the

149

tricyclic system. Although the biosynthetic pathway from shikimate to PDC is known, the

150

steps from PDC to myxin had not been completely established. With the heterologous

151

expression and purification of LaPhzM, LaPhzS and LaPhzNO1, we set out to reconstitute

152

the myxin biosynthetic pathway in vitro. PDC gave a retention time of 8.6 min on HPLC and

153

was converted to 1,6-dihydroxyphenazine (DHP, 7) by LaPhzS in the presence of the

154

cofactors FAD and NADH (Figure 4). DHP ran at 10.0 min and gave a m/z of 213.40 for [M

155

+ H]+ (Figure S4B). When the second enzyme LaPhzNO1 and another cofactor NADPH were

156

added in the reaction mixture, two new peaks appeared at 13.5 and 19.8 min, in addition to

157

DHP (Figure 4). The peak at 13.5 min co-migrated with 1,6-dihydroxyphenazine

158

N5-monooxide (DHPO, 8) on HPLC and gave a m/z of 229.08 for [M + H]+ (Figure S4H),

159

whereas the peak at 19.8 min showed the same retention time as iodinin

160

(1,6-dihydroxyphenazine N5,N10-dioxide).16 Finally, when the third enzyme LaPhzM and the

161

cofactor SAM were added to the reaction mixture, the peaks for PDC, DHP, DHPO, and

162

iodinin all disappeared. Instead, two new peaks at 9.2 and 10.3 min showed up on HPLC

163

(Figure 4). The peak at 10.3 min co-migrated with myxin on HPLC and gave a m/z of 259.39

164

for [M + H]+ (Figure 4 and Figure S4I), whereas the peak at 9.2 min gave a m/z of 273.26 for

165

[M + H]+, which is consistent with 1,6-dimethoxyphenazine N5,N10-dioxide (9) (Figure 4

166

and Figure S4J). This dimethoxy and dioxide phenazine is a new compound that had not been

167

observed in the previous studies of strain OH13.16 Together, the results demonstrate that three 8 ACS Paragon Plus Environment

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168

enzymes, LaPhzS, LaPhzNO1, and LaPhzM, are sufficient to decorate the shikimate-derived

169

PDC into the long-time used antibiotic myxin. In this pathway, the first step is the double

170

decarboxylative hydroxylation of PDC catalyzed by LaPhzS, which provides substrates for

171

both LaPhzNO1 and LaPhzM. The N-oxidation activity of LaPhzNO1 is relatively selective,

172

as it requires a free hydroxy group at carbon-1 or/and carbon-6 of the phenazine ring.16 In

173

contrast, the O-methylation activity of LaPhzM is relatively flexible. LaPhzM can use

174

non-oxide, mono-oxide, or di-oxide phenazines as substrates. This flexibility leads to a

175

variety of phenazine derivatives as found in strain OH13 (Figure 1).

176

Test of LaPhzS and LaPhzM for pyocyanin biosynthesis in vitro. In addition to

177

myxin, pyocyanin is another well-known phenazine natural product that has been subject to

178

many studies.1-4 Pyocyanin contains a methyl group at N5, but no O-methyl group (Figure 1).

179

LaPhzS and LaPhzM for myxin biosynthesis in L. antibioticus are the respective homologs of

180

PhzS and PhzM for pyocyanin biosynthesis in P. aeruginosa. LaPhzS and LaPhzM, together

181

with LaPhzNO1, are the decorating enzymes that covert the dicarboxylic PDC to the

182

antibiotic myxin, whereas PhzS and PhzM are the decorating enzymes that convert the

183

monocarboxylic PCA to the virulence factor pyocyanin.17-20 PhzM catalyzes the

184

N-methylation step in pyocyanin biosynthesis, producing the intermediate N5-methyl-PCA.

185

However, the N-methylated product has not been isolated. This is in contrast to the reactions

186

catalyzed by LaPhzM, which generated a variety of methylated products as described above.

187

One reason could be due to the unstable nature of N5-methyl-PCA, as suggested previously.18

188

On the other hand, there could be other possibilities. For example, PCA might not be the

189

direct substrate of PhzM; instead, 1-hydroxyphenazine (10), the product of PCA through a 9 ACS Paragon Plus Environment


Page 10 of 35

190

decarboxylative hydroxylation catalyzed by PhzS,17 could be the substrate of PhzM. This

191

hypothesis is in agreement with the previous observation that PhzM’s function required the

192

presence of PhzS in the reaction.18 To test if LaPhzM had any N-methylation activity, we

193

tested if the enzymes from strain OH13 were able to convert PCA to pyocyanin. When

194

LaPhzS was incubated with PCA in the presence of FAD and NADH, a small peak at 17.4

195

min was produced from PCA, which ran at 17.8 min on HPLC (Figure 5). This product gave

196

a m/z of 197.05 for [M + H]+, which is consistent with the mass of 1-hydroxyphenazine (10)

197

(Figure S4E). When both LaPhzS and LaPhzM were incubated with PCA in the presence of

198

FAD, NADH and SAM, another small peak at 17.1 min was produced, while the peak at 17.4

199

min disappeared on HPLC (Figure 5). This product gave a m/z of 211.03 for [M + H]+ (Figure

200

S4F), which is coincident with the mass of pyocyanin. However, none of the two small peaks

201

co-migrated with standard pyocyanin on HPLC, which had a retention time of 5.9 min

202

although it also gave a m/z of 211.09 for [M + H]+ (Figure 5 and Figure S4G). Therefore, the

203

product at 17.4 min is 1-hydroxyphenazine (10), and the product at 17.1 min is

204

1-methoxyphenazine (11). The results show that, like PhzS, LaPhzS is able to catalyze the

205

decarboxylative hydroxylation of PCA to produce 1-hydroxyphenazine, but LaPhzM can only

206

convert

207

1-hydroxy-N5-methylphenazine

208

N-methyltransferase when PCA is the substrate. Thus, LaPhzM is an O-methyltransferase,

209

whereas PhzM is an N-methyltransferase. Finally, it should be pointed out that only a small

210

fraction of PCA was converted to products 10 and 11 by LaPhzS and LaPhzM, suggesting

211

that the phenazine monocarboxylic acid is not an optimal substrate for the Lysobacter

1-hydroxyphenazine

to

1-methoxyphenazine,

(pyocyanin).

LaPhzM


does

not

rather function

than as

an

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212


enzymes.

213

Overview of the three-dimensional structure of LaPhzM. In order to shine light on

214

the structural basis of the enzyme activity and substrate selectivity of LaPhzM, we

215

determined a crystallographic structure of LaPhzM in complex with the cofactor at 1.42 Å,

216

using the mitomycin 7-O-methyltransferase MmcR from Streptomyces lavendulae as an

217

initial search model for phasing.23 An overview of the LaPhzM structure is shown in Figure

218

S6. Similar to many other O-methyltransferases, LaPhzM forms a dimeric structure with a

219

dimerization domain in the N-terminus and a catalytic domain with the α/β Rossmann fold in

220

the C-terminus of each monomer. The overall fold of LaPhzM mirrors that of MmcR despite

221

the low sequence identity between the two proteins (Figure S7A).23 All the secondary

222

structural elements are also conserved in PhzM (Figure S7B) with a larger structural variation

223

in comparison to that of MmcR.

224

The active site. The electron density around the cofactor binding site clearly indicates

225

an S-adenosyl-L-homocysteine (SAH), instead of a SAM, in the LaPhzM crystal structure

226

(Figure 6A). Since SAM was incubated with LaPhzM during crystallization, this observation

227

indicates that LaPhzM is capable of catalyzing the demethylation of SAM in the absence of

228

the phenazine substrate. The surrounding environment at the cofactor binding site of LaPhzM

229

closely resembles that of MmcR and other similar SAM-dependent O-methyltransferases

230

(Figure S8).23-24 The proteinaceous environment at the active site of LaPhzM possesses the

231

structural characteristics for the general acid/base-mediated transmethylation of Class I

232

O-methyltransferases.24 A 3D structural alignment shows that the His251-Glu306 pair takes

233

similar geometric arrangement as their corresponding residues (His259 and Glu313, 11 ACS Paragon Plus Environment


234

respectively) in MmcR (Figure 6B), which have been suggested to form a hydrogen bond

235

network and be involved in deprotonating the hydroxyl group of the substrate.23 Interestingly,

236

Asp252 (Asp260 in MmcR) adjacent to His251 and SAH is also within the hydrogen bonding

237

distance with the substrate near the active site. Sequence alignments indicate that His251,

238

Asp252 and Glu306 are highly conserved in the LaPhzM homologs and other similar

239

O-methyltransferases. It remains to be tested whether Asp252 plays a crucial role in

240

positioning the substrate or/and proton shuttling during the catalytic process.24

241

Modeling of the LaPhzM-substrate complex. Although the LaPhzM structure

242

determined in this study is in the substrate-free form, cavity analysis by CAVER and

243

structural comparison with the MmcR-SAH-mitomycin A complex (PDB ID: 3GXO) suggest

244

that the characterized LaPhzM-SAH complex is in an open state for substrate binding.23, 25

245

Indeed, 1-hydroxy-6-methoxyphenazine (6), the intermediate methylated product from DHP,

246

can be fit well into the substrate binding pocket of LaPhzM by ligand docking (Figure 7). In

247

this model, the substrate entry is surrounded by helices α9, α10 and α15 in one monomer and

248

is capped by helix α'1 from the second monomer, which leaves a narrow channel for substrate

249

entry (Figures 7A and 7B). At the substrate binding site, 6 is surrounded by eleven residues in

250

a largely hydrophobic environment (Figure 7C). Particularly, two aromatic residues Tyr158

251

and Phe290 located in helices α10 and α15, respectively, are at the surface of LaPhzM facing

252

the solvent environment, presumably serving as the gate of the substrate entry. These two

253

residues are conserved only in the close homologs of LaPhzM with high sequence identity

254

and likely play an important role in defining the substrate preference of LaPhzM. Tyr158 and

255

Phe290 are absent in MmcR, which leaves a wider opening gate to accommodate the large 12 ACS Paragon Plus Environment

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256

polar tail of mitomysin A. Apart from the hydrophobic and van der Waals interactions, and

257

the hydrogen bond network involving the three residues mentioned above at the active site, a

258

few polar residues including Gln103, His107, Gln155 and His297 may also form hydrogen

259

bonds with the 1-hydroxyl group, N5 and N10, respectively, in DHP and thus prepare the

260

substrate to the optimal orientation for transmethylation (Figure 7).

261

Overall, the local environment at the substrate binding pocket of LaPhzM strongly

262

suggests that proximity may also be an important mechanism for determining the enzymatic

263

activity and phenazine selectivity as observed in the enzymatic activity assay. The higher

264

activity of LaPhzM to 1,6-dihydroxyphenazine (DHP) in comparison to 1-hydroxyphenazine

265

is likely due to the hydrogen bonding interactions between one hydroxy group of DHP and

266

Glu103/His107 that facilitate the orientation of the second hydroxy optimal for methyl

267

transfer (Figure 7). Among the three phenazine substrates tested, LaPhzM shows the lowest

268

activity to 6-hydroxy-1-methoxyphenazine N5-oxide (2) (Figure S5). The proton in the

269

6-hydroxyl group of 2 has been proposed to form a stable hydrogen bond with oxidized N5 in

270

a pseudo six-member ring (Figure S9).12 The presence of an intramolecular hydrogen bond

271

network in 2 may reduce the efficiency of deprotonation on the 6-hydroxyl group mediated

272

by a hydrogen bonding with His251 and thus reduce the transmethylation rate.

273

Structural comparison of the substrate binding pocket in LaPhzM and PhzM. The

274

absence of the cofactor and substrate in the reported PhzM structure makes it difficult for a

275

direct comparison of the substrate binding pocket between the two structures. Nonetheless,

276

SAH can be modelled into the active site of PhzM using its homolog IOMT (PDB ID: 1FP2)

277

as a model, as demonstrated previously owing to the high structural similarity in this region.18 13 ACS Paragon Plus Environment


278

When overlaying the two structures around the SAH binding site, the most noticeable

279

difference is the solvent accessibility at the substrate binding pocket. The helices α9, α10 and

280

α15 in PhzM are further away from the active site (up to 7 Å relative to the corresponding Cα

281

atoms in LaPhzM). In addition, different from LaPhzM, helix α'1 in PhzM does not cover the

282

opening of the active site. These differences in the local structural arrangement result in a

283

much more solvent exposed active site in PhzM (Figure 8), which is not optimal for SAM

284

and/or substrate binding and the reaction of transmethylation. These observations may

285

explain the difficulties in co-crystalizing PhzM with a SAM/SAH.18

286

As discussed above in pyocyanin biosynthesis, it was proposed that failing to detect the

287

product when incubating PhzM with SAM and the substrate PCA in the absence of PhzS was

288

probably due to the instability of the product N5-methyl-PCA.18 However, no SAM

289

consumption was reported either in the study, while this would be expected if the

290

transmethylation did occur. The results from the structural comparison between PhzM and

291

LaPhzM provide structural insights in supporting that the alternative explanation may exist,

292

since a free PhzM as in the reported structure is not optimal for catalyzing the

293

transmethylation reaction. The interaction of PhzM with PhzS may be required for

294

desolvation of the active site, which is necessary for the stable cofactor/substrate binding and

295

efficient transmethylation. By interacting with PhzM, PhzS could either cap the substrate

296

binding pocket directly by itself, or trigger conformational changes in PhzM and lead to a

297

similar structural arrangement at the substrate binding pocket as observed in LaPhzM.

298

In conclusion, we have characterized the LaPhzM gene from strain OH13 and

299

demonstrated that LaPhzM is a SAM-dependent O-methyltransferase responsible for all 14 ACS Paragon Plus Environment

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300

methoxy formation in phenazine natural products isolated from strain OH13, including the

301

well-known antibiotic myxin. For the first time, we demonstrated the biosynthesis of myxin

302

from PDC, one of the two common precursors for all phenanzine natural products, through in

303

vitro reconstitution of the ring-decorating steps using purified enzymes. Finally, we

304

determined the cofactor-bound X-ray crystal structure of LaPhzM at 1.42 Å resolution, which

305

provides molecular insights into the catalytic activity and O-methyl selectivity of the enzyme.

306

These findings will facilitate future efforts to improve myxin production in strain OH13

307

through metabolic pathway engineering, and the identification of the phenazine-decorating

308

enzymes will find uses in chemoenzymatic syntheses of new phenazine antibiotics.

309 310

■ METHODS

311

312

Cloning and Expression of LaPhzM in E. coli. LaPhzM was amplified from the

313

genomic DNA of strain OH13 (CGMCC No.7561), using primers LaPhzM-PF/LaPhzM-PR

314

(Table S1). The product was cloned into the EcoRI/HindIII sites in plasmid pET28a

315

(Novagen) and confirmed by DNA sequencing. This construct was introduced into E. coli

316

BL21(DE3) for expression. LaPhzM was purified using a Ni-NTA column (GE Life Science)

317

and a size exclusion column (Sephadex 200) (Figure S2). A typical stock solution contained

318

LaPhzM of 30 mg/ml, in 50 mM Tris buffer pH 8, 200 mM NaCl and 1 mM DTT and was

319

stored at -80 °C. In the protein preparation for LaPhzM cyrstalization, the thrombin cleavge

320

site was removed from the LaPhzM expression construct.

321

Enzyme activity assays. Compound 2 was prepared from strain OH13 culture.16 PDC 15 ACS Paragon Plus Environment


322

and PCA were purchased from Alligator Reagent Inc., China, and pyocyanin from

323

Sigma-Aldrich. DHP (7) was purified by HPLC from the reaction mixture of PDC and

324

LaPhzS.16 The LaPhzS reaction was conducted in a 100 µl mixture containing LaPhzS (1

325

µM), the substrate (PDC or PCA, 100 µM), NADH (100 µM), and FAD (100 µM) in

326

phosphate buffer (KH2PO4/K2HPO4, 100 mM, pH 7.0). The reaction mixture was incubated

327

at room temperature for 30 min. The reaction was quenched with an equal volume of ethyl

328

acetate, and the organic phase containing the product was collected and evaporated to dryness.

329

The residues were dissolved in methanol (100 µl), and the supernatant was obtained by

330

centrifugation at 13,200 × g for 10 min. A fraction (20 µl) of the supernatant was subjected to

331

reverse-phase HPLC analysis or LC-MS as described previously.16 The LaPhzNO1-catalyzed

332

N-oxidation was carried out in the same conditions as that for LaPhzS, except NADH

333

replaced by NADPH (100 µM). The LaPhzM-catalyzed methyltransfer reaction was

334

conducted in a 100 µl mixture containing LaPhzM (5 µM), substrate (50 µM), SAM (180

335

µM), in Tris buffer (50 mM, pH 7.8). The mixture was incubated at room temperature for 30

336

min, and the reaction was stopped by adding ethyl acetate. To quantify the selectivity, kinetics

337

analysis was performed with compound 2 and SAM. Initial velocity of the reaction was

338

measured at a variety of substrate concentrations. The reaction was quantified by measuring

339

the SAM product, SAH, using reverse-phase HPLC (254 nm). A calibration curve of SAH

340

was prepared prior to the enzyme assays. These data were globally fitted to

341

Michaelis-Menten equation, yielding KM values. In the reactions to compare various potential

342

substrates for LaPhzM, the relative conversion rate was calculated based on the conversion

343

rate of compound 2 to compound 4, which was set as 100%. The relative rate of other 16 ACS Paragon Plus Environment

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344

substrates converted by LaPhzM was expressed as the percent of that of compound 2

345

converted to compound 4.

346

In reactions with PCA as substrate to test the activity of LaPhzS and LaPhzM in

347

pyocyanin biosynthesis in vitro, the procedures were the same as above, but with the

348

following modifications for HPLC analysis. A sample (20 µl) was injected to a

349

reversed-phase column (Cosmosil, 4.6 ID × 250 mm) in an Agilent HPLC (1220 Infinity LC).

350

Water/0.05% formic acid (solvent A) and methanol 0.05% formic acid (solvent B) were used

351

as the mobile phases with a flow rate of 1.0 ml/min. The HPLC program was as follows:

352

5−25% B in the first 5 min, 25-80% B in 5-25 min, 100% B at 26-35 min, back to 5% B at 36

353

min, and maintained to 40 min. The phenazines were detected at 254 nm on a UV detector.

354

LCQ-MS was used to verify the mass of the peak of PCA and products.

355

Crystallization. SAM (Biolab Inc) was added to the purified LaPhzM at a molar ratio of

356

1.5:1 before concentrating the protein to 20 mg/ml. LaPhzM was crystallized by sitting-drop

357

vapor diffusion at room temperature (~ 295 K). The protein solution at 20 mg/ml was mixed

358

in 1:1 volume ratio with the reservoir solution containing 0.2 M ammonium formate, 18 - 22 %

359

w/v PEG 3350. Crystals typically reached the maximum size in about one week. Santovac® 5

360

Cryo Oil (Hampton Research) was used as the cryoprotectant when harvesting the crystal.

361

X-ray Diffraction Data Collection, Processing and Refinement. X-ray diffraction

362

data were collected on Beamline 9-2 at the Stanford Synchrotron Radiation Lightsource

363

(SSRL), with a 6M Pixel Array Detector. A set of 1200 diffraction images was collected at

364

12,658 eV with an oscillation angle of 0.15°. Diffraction data were integrated with the XDS

365

program package,26 and subsequently processed using programs of the CCP4 suite.27 The 17 ACS Paragon Plus Environment


366

phase was determined by molecular replacement using structure of the mitomycin

367

7-O-methyltransferase MmcR from Streptomyces lavendulae (PDB code 3gwz, 34% of

368

sequence identity)23 as the search model and employing the BALBES automated molecular

369

replacement pipeline server.28 Further refinement and structural validation was carried out by

370

using REFMAC5, COOT and MolProbity.29-31 The diffraction data and refinement statistics

371

are listed in Tables S2 and S3, respectively. All but the 6xHis tag and the first 9 residues in

372

the N-terminus of the native protein have been included in the model. The programs COOT

373

and PyMOL (The PyMOL Molecular Graphics System, Version 1.7.1 Schrödinger) were used

374

for structural analysis and for generating figures. The atomic coordinates and experimental

375

data (code 6C5B) have been deposited in the Protein Data Bank (www.wwpdb.org).

376

Molecular Docking. The prediction of LaPhzM–substrate binding was performed with

377

Autodock Vina employing default parameters using a grid with dimensions of 26 × 20 × 20 Å

378

centered at the substrate binding cavity.32 Water molecules were removed from the LaPhzM

379

structure in preparation of docking. The coordinates for methylated phenazines were

380

modified from the structures downloaded from the PubChem database. The Gasteiger charges

381

and hydrogens were added to the receptor structure and phenazines with AutoDocktools.33

382

The receptor structure was treated as a rigid body during docking analysis to exclude the

383

uncertainty caused by the structural flexibilities.

384

385

■ ASSOCIATED CONTENT

386

Supporting Information

387

Details of primer sequences, statistics for data processing and refinement, multiple 18 ACS Paragon Plus Environment

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388

amino acid sequence alignment, SDS-PAGE of proteins, enzyme kinetics, protein secondary

389

structural elements, models of cofactor and substrate binding environments in protein

390

structures, and spectroscopic data for identification of phenazines. This material is available

391

free of charge via the Internet at http://pubs.acs.org.

392

393

■ AUTHOR INFORMATION

394

Corresponding Author:

395

*E-mail: [email protected]; Telephone: +402-4722998. Fax: +402-4729402

396

*E-mail: [email protected]; Telephone: +402-4722967. Fax: +402-4727842

397 398

Author Contributions:

399

L.D. and L.Z. conceived and designed experiments. J.J. and D.B. carried out experiments.

400

L.D., L.Z., J.J and A.S. analyzed data. J.J., L.D. and L.Z. wrote the manuscript.

401

402

■ ACKNOWLEDGMENTS

403

This work was supported in part by the NIH (R01AI097260), UNL Redox Biology

404

Center, a Faculty Seed Grant and a Revision Award from UNL. We thank Y. Zhao for helpful

405

discussion at the beginning of this project, R. Cerny and K. Wulser for technical assistance,

406

and H. Li for the insightful discussion on the charge distribution for molecular docking.

407

408 409



410

References

411

[1] Laursen, J. B., and Nielsen, J. (2004) Phenazine natural products: biosynthesis, synthetic

412 413 414

analogues, and biological activity. Chem. Rev. 104, 1663-1686. [2] Cimmino, A., Evidente, A., Mathieu, V., Andolfi, A., Lefranc, F., Kornienko, A., and Kiss, R. (2012) Phenazines and cancer. Nat. Prod. Rep. 29, 487-501.

415

[3] Mavrodi, D. V., Blankenfeldt, W., and Thomashow, L. S. (2006) Phenazine compounds in

416

fluorescent Pseudomonas spp. biosynthesis and regulation. Annu. Rev. Phytopathol.

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44, 417-445.

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[4] Mavrodi, D. V., Peever, T. L., Mavrodi, O. V., Parejko, J. A., Raaijmakers, J. M.,

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Lemanceau, P., Mazurier, S., Heide, L., Blankenfeldt, W., Weller, D. M., and

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Thomashow, L. S. (2010) Diversity and evolution of the phenazine biosynthesis

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pathway. Appl. Environ. Microbiol. 76, 866-879.

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[5] Pirnay, J. P., De Vos, D., Cochez, C., Bilocq, F., Vanderkelen, A., Zizi, M., Ghysels, B.,

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and Cornelis, P. (2002) Pseudomonas aeruginosa displays an epidemic population

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structure. Environ. Microbiol. 4, 898-911.

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[6] Selezska, K., Kazmierczak, M., Musken, M., Garbe, J., Schobert, M., Haussler, S.,

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Wiehlmann, L., Rohde, C., and Sikorski, J. (2012) Pseudomonas aeruginosa

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population structure revisited under environmental focus: impact of water quality and

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phage pressure. Environ. Microbiol. 14, 1952-1967.

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[7] Price-Whelan, A., Dietrich, L. E., and Newman, D. K. (2006) Rethinking 'secondary' metabolism: physiological roles for phenazine antibiotics. Nat. Chem. Biol. 2, 71-78. [8] Dietrich, L. E., Price-Whelan, A., Petersen, A., Whiteley, M., and Newman, D. K. (2006) 20 ACS Paragon Plus Environment

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The phenazine pyocyanin is a terminal signalling factor in the quorum sensing

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network of Pseudomonas aeruginosa. Mol. Microbiol. 61, 1308-1321.

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[9] Wang, Y., Wilks, J. C., Danhorn, T., Ramos, I., Croal, L., and Newman, D. K. (2011)

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Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron

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acquisition. J. Bacteriol. 193, 3606-3617.

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[10] Glasser, N. R., Kern, S. E., and Newman, D. K. (2014) Phenazine redox cycling

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enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of

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ATP and a proton-motive force. Mol. Microbiol. 92, 399-412.

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[11] Dietrich, L. E., Teal, T. K., Price-Whelan, A., and Newman, D. K. (2008) Redox-active

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antibiotics control gene expression and community behavior in divergent bacteria,

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Science 321, 1203-1206.

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[12] Chowdhury, G., Sarkar, U., Pullen, S., Wilson, W. R., Rajapakse, A., Fuchs-Knotts, T.,

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and Gates, K. S. (2012) DNA strand cleavage by the phenazine di-N-oxide natural

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product myxin under both aerobic and anaerobic conditions. Chem. Res. Toxicol. 25,

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197-206.

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[13] Costa, K. C., Glasser, N. R., Conway, S. J., and Newman, D. K. (2017) Pyocyanin

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degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa

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biofilms. Science 355, 170-173.

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[14] Pierson, L. S., 3rd, Gaffney, T., Lam, S., and Gong, F. (1995) Molecular analysis of

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genes encoding phenazine biosynthesis in the biological control bacterium

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Pseudomonas aureofaciens 30-84. FEMS Microbiol. Lett. 134, 299-307.

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[15] Mavrodi, D. V., Ksenzenko, V. N., Bonsall, R. F., Cook, R. J., Boronin, A. M., and 21 ACS Paragon Plus Environment


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Thomashow, L. S. (1998) A seven-gene locus for synthesis of phenazine-1-carboxylic

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acid by Pseudomonas fluorescens 2-79. J. Bacteriol. 180, 2541-2548.

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[16] Zhao, Y., Qian, G., Ye, Y., Wright, S., Chen, H., Shen, Y., Liu, F., and Du, L. (2016)

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Heterocyclic aromatic N-oxidation in the biosynthesis of phenazine antibiotics from

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Lysobacter antibioticus. Org. Lett. 18, 2495-2498.

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[17] Greenhagen, B. T., Shi, K., Robinson, H., Gamage, S., Bera, A. K., Ladner, J. E., and

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Parsons, J. F. (2008) Crystal structure of the pyocyanin biosynthetic protein PhzS.

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Biochemistry 47, 5281-5289.

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[18] Parsons, J. F., Greenhagen, B. T., Shi, K., Calabrese, K., Robinson, H., and Ladner, J. E.

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(2007) Structural and functional analysis of the pyocyanin biosynthetic protein PhzM

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from Pseudomonas aeruginosa. Biochemistry 46, 1821-1828.

465

[19] Gohain, N., Thomashow, L. S., Mavrodi, D. V., and Blankenfeldt, W. (2006) The

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purification,

crystallization

and

preliminary

structural

characterization

of

467

FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from

468

Pseudomonas aeruginosa. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62,

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989-992.

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[20] Gohain, N., Thomashow, L. S., Mavrodi, D. V., and Blankenfeldt, W. (2006) The

471

purification, crystallization and preliminary structural characterization of PhzM, a

472

phenazine-modifying methyltransferase from Pseudomonas aeruginosa. Acta

473

Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62, 887-890.

474

[21] Shen, B., and Hutchinson, C. R. (1993) Tetracenomycin F1 monooxygenase: oxidation

475

of a naphthacenone to a naphthacenequinone in the biosynthesis of tetracenomycin C 22 ACS Paragon Plus Environment

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in Streptomyces glaucescens. Biochemistry 32, 6656-6663.

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[22] Kendrew, S. G., Hopwood, D. A., and Marsh, E. N. (1997) Identification of a

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monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of

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actinorhodin: purification and characterization of the recombinant enzyme. J.

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Bacteriol. 179, 4305-4310.

481

[23] Singh, S., Chang, A., Goff, R. D., Bingman, C. A., Gruschow, S., Sherman, D. H.,

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Phillips, G. N., Jr., and Thorson, J. S. (2011) Structural characterization of the

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mitomycin 7-O-methyltransferase. Proteins 79, 2181-2188.

484

[24] Liscombe, D. K., Louie, G. V., and Noel, J. P. (2012) Architectures, mechanisms and

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molecular evolution of natural product methyltransferases. Nat. Prod. Rep. 29,

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1238-1250.

487

[25] Chovancova, E., Pavelka, A., Benes, P., Strnad, O., Brezovsky, J., Kozlikova, B., Gora,

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A., Sustr, V., Klvana, M., Medek, P., Biedermannova, L., Sochor, J., and Damborsky,

489

J. (2012) CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein

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structures. PLoS Comput. Biol. 8, e1002708.

491

[26] Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132.

492

[27] Collaborative Computational Project. (1994) The CCP4 suite: programs for protein

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crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760-763. [28] Long, F., Vagin, A. A., Young, P., and Murshudov, G. N. (2008) BALBES: a molecular-replacement pipeline. Acta Crystallogr. D Biol. Crystallogr. 64, 125-132.

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[29] Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of

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macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 23 ACS Paragon Plus Environment


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53, 240-255.

499

[30] Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray,

500

L. W., Arendall, W. B., 3rd, Snoeyink, J., Richardson, J. S., and Richardson, D. C.

501

(2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic

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acids. Nucleic Acids Res. 35, W375-383.

503 504

[31] Emsley, P., and Cowtan, K. (2004) COOT: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132.

505

[32] Trott, O., and Olson, A. J. (2010) AutoDock Vina: improving the speed and accuracy of

506

docking with a new scoring function, efficient optimization, and multithreading. J.

507

Comput. Chem. 31, 455-461.

508

[33] Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S.,

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and Olson, A. J. (2009) AutoDock4 and AutoDockTools4: Automated docking with

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selective receptor flexibility. J. Comput. Chem. 30, 2785-2791.

511 512


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513

Figure 1. Chemical structure of selected phenazine natural products isolated from

514

Pseudomonas aeruginosa and Lysobacter antibioticus. Some of the functional groups on the

515

phenazine ring are color highlighted to indicate the decorations relevant to this research.

516

Figure

517

6-hydroxy-1-methoxyphenazine N5-oxide (2) as substrate. (A) LaPhzM + substrate with

518

SAM.

519

Figure 3. HPLC analysis of reaction products of LaPhzM with 1,6-dihydroxyphenazine

520

(DHP, 7) as substrate. From top to bottom, the HPLC traces are for reactions incubated for 0,

521

15, 45, 90 min, respectively.

522

Figure 4. In vitro reconstitution of myxin biosynthesis. In addition to myxin and other known

523

phenazines, the reactions produced a new compound (9), a phenazine with dimethoxy and

524

di-N-oxide, which had not been isolated from strain OH13.

525

Figure 5. HPLC analysis of reaction products of LaPhzS and LaPhzM with PCA as substrate.

526

Pyocyanin was also included as a reference.

527

Figure 6. The active site of the LaPhzM. (A) The 2Fo-Fc electron density map around the

528

cofactor binding site. The map is illustrated in light blue color and contoured at 3σ. The

529

cofactor product S-adenosyl-L-homocysteine (SAH) is represented by sticks. (B) The stick

530

representation of the active site in LaPhzM (cyan carbon atoms) overlaid with the

531

MmcR-SAH-mitomycin A (MMA) complex (PDB ID: 3GXO, gray carbon atoms). The

532

residues in LaPhzM are labeled in cyan fonts and the corresponding ones in MmcR are

533

labeled in black fonts in the round bracket. The nitrogen atoms are in blue, oxygen in red and

534

sulfur in yellow.

2.

HPLC

analysis

of

the

reaction

product

of

LaPhzM

with

(B) Boiled LaPhzM + substrate with SAM. (C) Substrate 2.



535

Figure 7. The substrate binding pocket of LaPhzM with 6 fit into the structure by

536

autodocking. (A) and (B) are the surface representations of LaPhzM viewed from the

537

protein-solvent interface toward the substrate binding pocket with and without the second

538

monomer of the dimer, respectively. The black arrow indicates the proposed narrow substrate

539

entry tunnel between helices α9, α10 and α15 from one monomer and helix α'1 from the

540

second monomer. The surface presentation is colored by the underlying atoms with nitrogen

541

in blue and oxygen in red. The surface around carbon atoms is colored white for one monoer,

542

and yellow for the second promoter for clarity. The local environment at the substrate binding

543

pocket is shown in (C). The carbon atoms are colored cyan for the amino acid residues and

544

gray for SAH and 6. The water molecule is shown in the red sphere. SAH, 6 and the amino

545

acid residues within 4 Å of 6 are represented by sticks.

546

Figure 8. Comparison of the substrate binding pocket of PhzM (PDB ID: 2IP2) and LaPhzM.

547

(A) A ribbon presentation of PhzM (blue) overlaid with LaPhzM (light gray). Only the

548

residues from AA121 to the end of the C-terminus are included for clarity. (B) A surface

549

presentation around the active site of PhzM viewed from the protein-solvent interface. The

550

surface presentation is colored by the underlying atoms with nitrogen in blue and oxygen in

551

red. The surface around carbon atoms is colored white for one monomer and yellow for the

552

second promoter for clarity. SAH was modelled to the active site of PhzM guided by its

553

homolog IOMT (PDB ID: 1FP2); 5-Me-PCA (N5-methylphenazine carboxylic acid) was

554

manually placed near to SAH for the purpose of demonstration.


Page 26 of 35

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555


Figure 1. 9 8 7 6

N

10a

5a

N

4a

5

1 2

N

N

N

N

Phenazine

O

Phenazine 1-carboxylic acid (PCA)

Pyocyanin

O

OH

O

OH

N+

1 (Myxin)

OH

OH

O

OH

N+

N OH

N OH

7 (DHP)

O

8 (DHPO)

OCH3

OCH3 N

N

OCH3

O

OH

N

N

O

R

CH3

N+

3 (Iodinin)

2

N

OH

N+

N OCH3

OCH3 O

N

Phenazine Aeruginosin A (R=H) 1,6-dicarboxylic Aeruginosin B (R=SO3H) acid (PDC)

N+

N+

N+

H2N

N COOH

CH3

556

N

N

3 4

COO

COOH

COOH

O

10 9a

N

OCH3

OCH3

6

5

4

OH

OCH3

OCH3

N+

N

N

N+

N

N

10

11

OCH3 O

9

557 558

Figure 1. Chemical structure of selected phenazine natural products isolated from

559

Pseudomonas aeruginosa and Lysobacter antibioticus. Some of the functional groups on the

560

phenazine ring are color highlighted to indicate the decorations relevant to this research.

561 562 563 564



565

Figure 2. O-

O-

OH

N+

SAM

OCH3

O CH3

N+

LaPhzM

N

566

Page 28 of 35

N OCH 3

567 568

2.

HPLC

analysis

of

the

reaction

product

of

LaPhzM

with

569

Figure

570

6-hydroxy-1-methoxyphenazine N5-oxide (2) as substrate. (A) LaPhzM + substrate with

571

SAM.

(B) Boiled LaPhzM + substrate with SAM. (C) Substrate 2.

572 573 574 575 576


Page 29 of 35 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

577


Figure 3.

578 579

580 581 582

Figure 3. HPLC analysis of reaction products of LaPhzM with 1,6-dihydroxyphenazine

583

(DHP, 7) as substrate. From top to bottom, the HPLC traces are for reactions incubated for 0,

584

15, 45, 90 min, respectively.

585 586 587 588 589 590 591



592

Page 30 of 35

Figure 4. COOH N

N COOH

PDC

OH

LaPhzS

N

O2 NADH

N OH

O

OH

O

LaPhzNO1

N

LaPhzNO1

O2 NADPH

N

O2 NADPH

DHP (7)

OH

N OH

DHPO (8)

O

Iodinin (3) SAM

LaPhzM O

LaPhzM

N

SAM

9

593

O

O CH3

N

OCH 3 O

OH

N

OH

N

N OCH 3 O

Myxin (1)

594 595 596

Figure 4. In vitro reconstitution of myxin biosynthesis. In addition to myxin and other known

597

phenazines, the in vitro reactions produced a new compound (9), a phenazine with dimethoxy

598

and di-N-oxide, which had not been isolated from strain OH13.

599


Page 31 of 35 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

600 601


Figure 5.

602

603 604

Figure 5. HPLC analysis of reaction products of LaPhzS and LaPhzM with PCA as substrate.

605

Pyocyanin was also included as a reference.

606



607

Figure 6.

608 609

Figure 6. The active site of the LaPhzM. (A) The 2Fo-Fc electron density map around the

610

cofactor binding site. The map is illustrated in light blue color and contoured at 3σ. The

611

cofactor product S-adenosyl-L-homocysteine (SAH) is represented by sticks. (B) The stick

612

representation of the active site in LaPhzM (cyan carbon atoms) overlaid with the

613

MmcR-SAH-mitomycin A (MMA) complex (PDB ID: 3GXO, gray carbon atoms). The

614

residues in LaPhzM are labeled in cyan fonts and the corresponding ones in MmcR are

615

labeled in black fonts in the round bracket. The nitrogen atoms are in blue, oxygen in red and

616

sulfur in yellow.

617 618


Page 32 of 35

Page 33 of 35 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

619


Figure 7. C

A

α10

α15

D252

SAH

H251 E306

α9 F151

M298

6 L302

B Q155

L294 Q111

H297 Y158

α'1

620

HOH1

F290 H107

Q103

621

Figure 7. The substrate binding pocket of LaPhzM with 6 fit into the structure by

622

autodocking. (A) and (B) are the surface representations of LaPhzM viewed from the

623

protein-solvent interface toward the substrate binding pocket with and without the second

624

monomer of the dimer, respectively. The black arrow indicates the proposed narrow substrate

625

entry tunnel between helices α9, α10 and α15 from one monomer and helix α'1 from the

626

second monomer. The surface presentation is colored by the underlying atoms with nitrogen

627

in blue and oxygen in red. The surface around carbon atoms is colored white for one

628

monomer, and yellow for the second monomer for clarity. The local environment at the

629

substrate binding pocket is shown in (C). The carbon atoms are colored cyan for the amino

630

acid residues and gray for SAH and 6. The water molecule is shown in the red sphere. SAH,

631

6 and the amino acid residues within 4 Å of 6 are represented by sticks.

632 633



634

Figure 8.

A

B

α10

α15 α9

5-Me-PCA

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

Page 34 of 35

SAH

α8

635 636 637

Figure 8. Comparison of the substrate binding pocket of PhzM (PDB ID: 2IP2) and LaPhzM.

638

(A) A ribbon presentation of PhzM (blue) overlaid with LaPhzM (light gray). Only the

639

residues from AA121 to the end of the C-terminus are included for clarity. (B) A surface

640

presentation around the active site of PhzM viewed from the protein-solvent interface. The

641

surface presentation is colored by the underlying atoms with nitrogen in blue and oxygen in

642

red. The surface around carbon atoms is colored white for one monomer and yellow for the

643

second monomer for clarity. SAH was modelled to the active site of PhzM guided by its

644

homolog IOMT (PDB ID: 1FP2); 5-Me-PCA (N5-methylphenazine carboxylic acid) was

645

manually placed near to SAH for the purpose of demonstration.

646


Page 35 of 35 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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Methyltransferase LaPhzM f - ACS Publications - American Chemical

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