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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
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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
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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
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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
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facilitate future exploitation of the thousands phenazines as new antibiotics through
35
metabolic engineering and chemoenzymatic syntheses.
and
aromatic
N-oxidations
of
PDC
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to
produce
iodinin
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Phenazines are a large group of heterotricyclic N-containing aromatic compounds. Over
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6000 phenazines have been isolated from living organisms or chemically synthesized during
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the last one and half century.1-4 Among them, several hundreds were reported to possess
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biological activities, including antimicrobial, antimalarial, and anticancer functions. The first
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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
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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
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the ring system. The decorating groups, including carboxylate, hydroxy, amide, N-methyl,
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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
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modified by surrounding organisms as a means to control the pathogen’s biofilms.13 The
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biosynthetic mechanism for the phenazine core has been extensively investigated by several
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groups.1-4,
53
1-carboxylic acid (PCA) and/or phenazine-1,6-dicarboxylic acid (PDC) (Figure 1). In
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contrast, relatively little has been done on the molecular mechanism underlying the
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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
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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
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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
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well-known antibiotic myxin (1-hydroxy-6-methoxyphenazine- N5,N10-dioxide).16 Myxin
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possesses potent antimicrobial activity with a unique mode of action and was manufactured
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as a copper (II) complex, Cuprimyxin, which had been used for decades as a topical broad
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spectrum veterinary antibiotic and antifungal drug.12 Five of the six compounds isolated from
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L. antibioticus contain one or two O-methyl groups, but no N-methyl group (Figure 1).16 The
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O-methylation is important for the antibiotic activity of phenazines. For example, myxin and
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iodinin (1,6-dihydroxyphenazine N5,N10-dioxide) have an identical structure, except that
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none of the hydroxy groups is methylated in iodinin (Figure 1), but myxin has much higher
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antimicrobial activity than iodinin in all the tests we conducted.16 While O-methylation is
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very common in natural product biosynthesis, the O-methylation in myxin and other
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phenazines had not been investigated.
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In pyocyanin, two enzymes are involved in the decoration of the phenazine ring during
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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
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N-methylation product, N5-methyl-PCA, was not isolated in the PhzM reaction. It was
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proposed that N5-methyl-PCA could be unstable and served as an enzyme-bound
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intermediate that was further processed by the second enzyme, PhzS, to produce the final
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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
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production of pyocyanin by preventing the formation and release of the unstable intermediate
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N5-methyl-PCA. The crystal structure of both PhzM and PhzS had been reported.17-20 The 1.8
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Å dimeric structure of PhzM showed three domains per monomer characteristic of many
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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.
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Phenazine ring decorations are usually specific to each producing organism. In strain
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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
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catalyzes twice of decarboxylative hydroxylation of PDC, the common precursor of all
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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
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N-oxidation at both N5 and N10 of the phenazine core. This enzyme eventually converts
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1,6-dihydroxyphenazine (7) to 1,6-dihydroxyphenazine N5,N10-dioxide (iodinin, 3). Within
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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
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small monooxygenases that catalyze oxidation of phenolic compounds to corresponding
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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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from strain OH13. We characterized the SAM-dependent O-methyltransferase LaPhzM
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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
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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
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phenazine biosynthetic gene cluster LaPhz in strain OH13 was heterologously expressed in E.
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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
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at the retention time of 14.3 min on HPLC, and the reaction yielded one new product with the
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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
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produced this compound. The new product had the same retention time as
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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
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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
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calculated mass of the compound (calculated mass 212.06). After 15 min of reaction, a major
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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
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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
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dihydroxy-containing
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monohydroxy-containing phenazine (10), monohydroxy-monomethoxy-containing phenazine
phenazine
DHP
(7)
was
the
7 ACS Paragon Plus Environment
substrate,
followed
by
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(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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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
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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
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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
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does
not
rather function
than as
an
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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
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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,
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SAH can be modelled into the active site of PhzM using its homolog IOMT (PDB ID: 1FP2)
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as a model, as demonstrated previously owing to the high structural similarity in this region.18 13 ACS Paragon Plus Environment
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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.
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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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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
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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.
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Enzyme activity assays. Compound 2 was prepared from strain OH13 culture.16 PDC 15 ACS Paragon Plus Environment
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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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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.
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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.
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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
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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
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uncertainty caused by the structural flexibilities.
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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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amino acid sequence alignment, SDS-PAGE of proteins, enzyme kinetics, protein secondary
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structural elements, models of cofactor and substrate binding environments in protein
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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
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References
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[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.
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[3] Mavrodi, D. V., Blankenfeldt, W., and Thomashow, L. S. (2006) Phenazine compounds in
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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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[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.
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[19] Gohain, N., Thomashow, L. S., Mavrodi, D. V., and Blankenfeldt, W. (2006) The
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purification,
crystallization
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preliminary
structural
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of
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FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from
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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
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purification, crystallization and preliminary structural characterization of PhzM, a
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phenazine-modifying methyltransferase from Pseudomonas aeruginosa. Acta
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Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62, 887-890.
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[21] Shen, B., and Hutchinson, C. R. (1993) Tetracenomycin F1 monooxygenase: oxidation
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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.
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[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.
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[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.
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[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,
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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.
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[26] Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132.
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[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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53, 240-255.
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[30] Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray,
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L. W., Arendall, W. B., 3rd, Snoeyink, J., Richardson, J. S., and Richardson, D. C.
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(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
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docking with a new scoring function, efficient optimization, and multithreading. J.
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Comput. Chem. 31, 455-461.
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[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.
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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.
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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.
26 ACS Paragon Plus Environment
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555
ACS Chemical Biology
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
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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
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577
ACS Chemical Biology
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
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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
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600 601
ACS Chemical Biology
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
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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
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619
ACS Chemical Biology
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
33 ACS Paragon Plus Environment
ACS Chemical Biology
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
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ACS Chemical Biology
TOC Graph 47x15mm (600 x 600 DPI)
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