Functional and Structural Analysis of Phenazine O -Methyltransferase LaPhzM from Lysobacter antibioticus OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin

Article abstract of DOI:10.1021/acschembio.8b00062

Myxin is a well-known antibiotic that had been used for decades. It belongs to the phenazine natural products that exhibit various biological activities, which are often dictated by the decorating groups on the heteroaromatic three-ring system. The three rings of myxin carry a number of decorations, including an unusual aromatic N5,N10-dioxide. We previously showed that phenazine 1,6-dicarboxylic acid (PDC) is the direct precursor of myxin, and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative hydroxylation and aromatic N-oxidations of PDC to produce iodinin (1.6-dihydroxy-N5,N10-dioxide phenazine). In this work, we identified the LaPhzM gene from Lysobacter antibioticus OH13 and demonstrated that LaPhzM encodes a SAM-dependent O-methyltransferase converting iodinin to myxin. The results further showed that LaPhzM is responsible for both monomethoxy and dimethoxy formation in all phenazine compounds isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing O-methylation of phenazines with non-, mono-, or di-N-oxide. In addition, we demonstrated a one-pot biosynthesis of myxin by in vitro reconstitution of the three phenazine-ring decorating enzymes. Finally, we determined the X-ray crystal structure of LaPhzM with a bound cofactor at 1.4 ? resolution. The structure provided molecular insights into the activity and selectivity of the first characterized phenazine O-methyltransferase. These results will facilitate future exploitation of the thousands of phenazines as new antibiotics through metabolic engineering and chemoenzymatic syntheses.

Full text of DOI:10.1021/acschembio.8b00062

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Functional and Structural Analysis of Phenazine O-
Methyltransferase 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
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Functional and Structural Analysis of Phenazine OꢀMethyltransferase LaPhzM from
Lysobacter antibioticus OH13 and OneꢀPot Enzymatic Synthesis of the Antibiotic Myxin
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Jiasong Jiang,† Daisy Guiza Beltran,‡ Andrew Schacht,‡ Stephen Wright,† Limei
Zhang,*,‡,§ and Liangcheng Du*,†
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†Departments of Chemistry, University of NebraskaꢀLincoln, NE 68588, USA
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‡Departments of Biochemistry, §Redox Biology Center and the Nebraska Center for
Integrated Biomolecular Communication, University of NebraskaꢀLincoln, NE 68588, USA
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Note: JJ and DGB contributed equally.
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ABSTRACT: Myxin is a wellꢀknown antibiotic that had been used for decades. It
belongs to the phenazine natural products that exhibit various biological activities, which are
often dictated by the decorating groups on the heteroaromatic threeꢀring system. The three
rings of myxin carry a number of decorations, including an unusual aromatic N5,N10ꢀdioxide.
We previously showed that phenazine 1,6ꢀdicarboxylic acid (PDC) is the direct precursor of
myxin and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative
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hydroxylation
and
aromatic
Nꢀoxidations
of
PDC
to
produce
iodinin
(1.6ꢀdihydroxyꢀN5,N10ꢀdioxide phenazine). In this work, we identified LaPhzM gene from
Lysobacter antibioticus OH13 and demonstrated that LaPhzM encodes a SAMꢀdependent
Oꢀmethyltransferase converting iodinin to myxin. The results further showed that LaPhzM is
responsible for both monoꢀmethoxy and diꢀmethoxy formation in all phenazine compounds
isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing
Oꢀmethylation of phenazines with nonꢀ, monoꢀ, or diꢀNꢀoxide. In addition, we demonstrated a
oneꢀpot biosynthesis of myxin by in vitro reconstitution of the three phenazineꢀring
decorating enzymes. Finally, we determined the Xꢀray crystal structure of LaPhzM with a
bound cofactor at 1.4 Å resolution. The structure provided molecular insights into the activity
and selectivity of the first characterized phenazine Oꢀmethyltransferase. These results will
facilitate future exploitation of the thousands phenazines as new antibiotics through
metabolic engineering and chemoenzymatic syntheses.
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Phenazines are a large group of heterotricyclic Nꢀcontaining aromatic compounds. Over
6000 phenazines have been isolated from living organisms or chemically synthesized during
the last one and half century.1ꢀ4 Among them, several hundreds were reported to possess
biological activities, including antimicrobial, antimalarial, and anticancer functions. The first
phenazine, pyocyanin (1ꢀhydroxyꢀN5ꢀmethylphenazine), was isolated from the Gram
negative opportunistic pathogen Pseudomonas aeruginosa in the late 19th century (Figure
1).1 This bacterial pathogen uses pyocyanin as a virulence factor for infection,5ꢀ6 and recent
work has showed that pyocyanin is a redoxꢀactive metabolite facilitating extracellular
electron transfer and survival in anoxic environments, which is critical for biofilm
development.7ꢀ11
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All phenazines share a threeꢀring core (dibenzopyrazine) but differ at the decorations on
the ring system. The decorating groups, including carboxylate, hydroxy, amide, Nꢀmethyl,
Oꢀmethyl (methyoxy), and prenyl groups, dictate the difference in phenazine biological
activities.12 Recent studies also showed that phenazines of P. aeruginosa could be selectively
modified by surrounding organisms as a means to control the pathogen’s biofilms.13 The
biosynthetic mechanism for the phenazine core has been extensively investigated by several
groups.1ꢀ4, 14ꢀ15 Five to seven genes are required to convert chorismic acid to phenazine
1ꢀcarboxylic acid (PCA) and/or phenazineꢀ1,6ꢀdicarboxylic acid (PDC) (Figure 1). In
contrast, relatively little has been done on the molecular mechanism underlying the
decorations on the rings. Phenazine natural products with a hydroxy group at the six
modifiable carbons of the phenazine core have been isolated from Pseudomonas species.1
Interestingly, the hydroxy groups are rarely methylated, although several wellꢀknown
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Pseudomonas products contain an Nꢀmethyl group, such as pyocyanin and aeruginosins
(Figure 1). In L. antibioticus OH13, we previously isolated six phenazines (1ꢀ6) including the
wellꢀknown antibiotic myxin (1ꢀhydroxyꢀ6ꢀmethoxyphenazineꢀ N5,N10ꢀdioxide).16 Myxin
possesses potent antimicrobial activity with a unique mode of action and was manufactured
as a copper (II) complex, Cuprimyxin, which had been used for decades as a topical broad
spectrum veterinary antibiotic and antifungal drug.12 Five of the six compounds isolated from
L. antibioticus contain one or two Oꢀmethyl groups, but no Nꢀmethyl group (Figure 1).16 The
Oꢀmethylation is important for the antibiotic activity of phenazines. For example, myxin and
iodinin (1,6ꢀdihydroxyphenazine N5,N10ꢀdioxide) have an identical structure, except that
none of the hydroxy groups is methylated in iodinin (Figure 1), but myxin has much higher
antimicrobial activity than iodinin in all the tests we conducted.16 While Oꢀmethylation is
very common in natural product biosynthesis, the Oꢀmethylation in myxin and other
phenazines had not been investigated.
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In pyocyanin, two enzymes are involved in the decoration of the phenazine ring during
biosynthesis in P. aeruginosa.17ꢀ20 The first enzyme (PhzM) is an Sꢀadenosylmethionine
(SAM) dependent Nꢀmethyltransferase, converting PCA to N5ꢀmethylꢀPCA. Curiously, PhzM
was shown to be active only in the physical presence of PhzS, a flavinꢀdependent
hydroxylase catalyzing a decaroxylative hydroxylation in pyocyanin biosynthesis. The
Nꢀmethylation product, N5ꢀmethylꢀPCA, was not isolated in the PhzM reaction. It was
proposed that N5ꢀmethylꢀPCA could be unstable and served as an enzymeꢀbound
intermediate that was further processed by the second enzyme, PhzS, to produce the final
product, pyocyanin. The result suggested that a proteinꢀprotein interaction between PhzM and
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PhzS might be required in pyocyanin production, as this interaction could ensure an efficient
production of pyocyanin by preventing the formation and release of the unstable intermediate
N5ꢀmethylꢀPCA. The crystal structure of both PhzM and PhzS had been reported.17ꢀ20 The 1.8
Å dimeric structure of PhzM showed three domains per monomer characteristic of many
small molecule methyltransferase but did not contain any ligand. The 2.4 Å crystal structure
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
OH13, the LaPhz gene cluster contains four genes that were predicted to be responsible for
phenazine ring decoration.16 One gene codes for the FADꢀdependent enzyme LaPhzS that
catalyzes twice of decarboxylative hydroxylation of PDC, the common precursor of all
phenazines in strain OH13 (Figure 1). This reaction converts PDC to 1,6ꢀdihydroxyphenazine
(7). The next gene encodes LaPhzNO1, a BaeyerꢀVilligerꢀlike flavin enzyme that catalyzes
Nꢀoxidation at both N5 and N10 of the phenazine core. This enzyme eventually converts
1,6ꢀdihydroxyphenazine (7) to 1,6ꢀdihydroxyphenazine N5,N10ꢀdioxide (iodinin, 3). Within
the LaPhz gene cluster, there remain two genes (LaPhzM and LaPhzX) that have not been
investigated for their function. LaPhzM shows a sequence similarity to genes coding for
methyltransferases including pyocyanin PhzM,18 and LaPhzX is similar to genes encoding
small monooxygenases that catalyze oxidation of phenolic compounds to corresponding
quinones and do not require any metal ion or other cofactors.21ꢀ22 Interestingly, although
LaPhzM is homologous to pyocyanin PhzM (Figure S1), no Nꢀmethyl containing phenazines
have been identified from strain OH13. In this work, we showed that LaPhzM is responsible
for Oꢀmethylation in the biosynthesis of myxin and other phenazine natural products isolated
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from strain OH13. We characterized the SAMꢀdependent Oꢀmethyltransferase LaPhzM
through heterologous expression, in vitro assay of the enzyme activity and selectivity, oneꢀpot
reconstitution of myxin biosynthesis in vitro, and determination of the crystal structure of
LaPhzM complexed with cofactor.
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107 ■ RESULTS AND DISCUSSION
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LaPhzM encodes an Oꢀmethyltransferase for production of both monoꢀmethylated
and diꢀmethylated phenazines in L. antibioticus OH13. The LaPhzM gene within the
phenazine biosynthetic gene cluster LaPhz in strain OH13 was heterologously expressed in E.
coli as a 36.7 kDa protein (Figure S2). To study the role of LaPhzM in the biosynthesis of
myxin and other phenazines, we incubated LaPhzM with 6ꢀhydroxyꢀ1ꢀmethoxyphenazine
N5ꢀoxide (2), the main phenazine compound produced by strain OH13.16 The substrate eluted
at the retention time of 14.3 min on HPLC, and the reaction yielded one new product with the
retention time of 6.8 min (Figure 2 and Figure S3). The production of this product was
dependent on LaPhzM and SAM, as neither the boiled LaPhzM nor the lack of SAM
produced this compound. The new product had the same retention time as
1,6ꢀdimethoxyphenazine N5ꢀoxide (4), which had previously been isolated from strain
OH13.16 Mass spectrometry analysis of the new product gave a m/z of 257.07 (Figure S4A),
which is also consistent with [M + H]+ for 1,6ꢀdimethoxyphenazine N5ꢀoxide (calculated
mass 256.08). The result demonstrates that LaPhzM is
a
SAMꢀdependent
Oꢀmethyltransferase for phenazines. Kinetics study of the enzyme gave a KM of 33.6 ꢁM and
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kcat of 0.6 minꢀ1, with Vmax of 2.9 ꢁM minꢀ1, when 6ꢀhydroxyꢀ1ꢀmethoxyphenazine N5ꢀoxide
(2) was the substrate to produce 1,6ꢀdimethoxyphenazine N5ꢀoxide (4) (Figure S3).
In addition to compounds 2 and 4, strain OH13 produces three other Oꢀmethyl
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containing
phenazines,
including
1ꢀhydroxyꢀ6ꢀmethoxyphenazine
(6),
1,6ꢀdimethoxyphenazine (5), and myxin (1).16 To further understand the role and substrate
selectivity of LaPhzM, we tested the enzyme’s activity with dihydroxyl phenazine as
substrate (Figure 3). 1,6ꢀDihydroxyphenazine (DHP, 7) gave the retention time of 10.1 min
and a m/z of 213.40 for [M + H]+ (Figure 3 and Figure S4B), which is also consistent with the
calculated mass of the compound (calculated mass 212.06). After 15 min of reaction, a major
peak at 9.8 min appeared. This product coꢀmigrated with 1ꢀhydroxyꢀ6ꢀmethoxyphenazin (6)
and gave a m/z of 227.02 for [M + H]+ (Figure 3 and Figure S4C), which is consistent with
the calculated mass of 6 (calculated mass 226.07). As the reaction continued, the peaks for
the substrate (7) and the first product (6) decreased and a third peak at 9.5 min became the
predominate product in reaction mixture (Figure 3). This peak had the same retention time as
1,6ꢀdimethoxyphenazine (5) and gave a m/z of 241.02 for [M + H]+ (Figure S4D), which is
consistent with the calculated mass of 5 (calculated mass 240.09). The results show that
LaPhzM is responsible for Oꢀmethylation of both the monoꢀmethylated and diꢀmethylated
phenazines produced by strain OH13.16
Additionally, we compared the relative reaction rate of LaPhzM toward different
substrates (Figure S5). The enzyme exhibited the highest relative activity when the
dihydroxyꢀcontaining phenazine DHP (7) was the substrate, followed by
monohydroxyꢀcontaining phenazine (10), monohydroxyꢀmonomethoxyꢀcontaining phenazine
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(2), and nonꢀphenazine substrates (8ꢀhydroxyquinoline and 6ꢀhydroxyquinoline).
Reconstitution of myxin biosynthesis in vitro. Myxin is perhaps the most interesting
phenazine natural product due to its potent antibiotic activity and rich decorations on the
tricyclic system. Although the biosynthetic pathway from shikimate to PDC is known, the
steps from PDC to myxin had not been completely established. With the heterologous
expression and purification of LaPhzM, LaPhzS and LaPhzNO1, we set out to reconstitute
the myxin biosynthetic pathway in vitro. PDC gave a retention time of 8.6 min on HPLC and
was converted to 1,6ꢀdihydroxyphenazine (DHP, 7) by LaPhzS in the presence of the
cofactors FAD and NADH (Figure 4). DHP ran at 10.0 min and gave a m/z of 213.40 for [M
+ H]+ (Figure S4B). When the second enzyme LaPhzNO1 and another cofactor NADPH were
added in the reaction mixture, two new peaks appeared at 13.5 and 19.8 min, in addition to
DHP (Figure 4). The peak at 13.5 min coꢀmigrated with 1,6ꢀdihydroxyphenazine
N5ꢀmonooxide (DHPO, 8) on HPLC and gave a m/z of 229.08 for [M + H]+ (Figure S4H),
whereas the peak at 19.8 min showed the same retention time as iodinin
(1,6ꢀdihydroxyphenazine N5,N10ꢀdioxide).16 Finally, when the third enzyme LaPhzM and the
cofactor SAM were added to the reaction mixture, the peaks for PDC, DHP, DHPO, and
iodinin all disappeared. Instead, two new peaks at 9.2 and 10.3 min showed up on HPLC
(Figure 4). The peak at 10.3 min coꢀmigrated with myxin on HPLC and gave a m/z of 259.39
for [M + H]+ (Figure 4 and Figure S4I), whereas the peak at 9.2 min gave a m/z of 273.26 for
[M + H]+, which is consistent with 1,6ꢀdimethoxyphenazine N5,N10ꢀdioxide (9) (Figure 4
and Figure S4J). This dimethoxy and dioxide phenazine is a new compound that had not been
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observed in the previous studies of strain OH13.16 Together, the results demonstrate that three
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enzymes, LaPhzS, LaPhzNO1, and LaPhzM, are sufficient to decorate the shikimateꢀderived
PDC into the longꢀtime used antibiotic myxin. In this pathway, the first step is the double
decarboxylative hydroxylation of PDC catalyzed by LaPhzS, which provides substrates for
both LaPhzNO1 and LaPhzM. The Nꢀoxidation activity of LaPhzNO1 is relatively selective,
as it requires a free hydroxy group at carbonꢀ1 or/and carbonꢀ6 of the phenazine ring.16 In
contrast, the Oꢀmethylation activity of LaPhzM is relatively flexible. LaPhzM can use
nonꢀoxide, monoꢀoxide, or diꢀoxide phenazines as substrates. This flexibility leads to a
variety of phenazine derivatives as found in strain OH13 (Figure 1).
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Test of LaPhzS and LaPhzM for pyocyanin biosynthesis in vitro. In addition to
myxin, pyocyanin is another wellꢀknown phenazine natural product that has been subject to
many studies.1ꢀ4 Pyocyanin contains a methyl group at N5, but no Oꢀmethyl group (Figure 1).
LaPhzS and LaPhzM for myxin biosynthesis in L. antibioticus are the respective homologs of
PhzS and PhzM for pyocyanin biosynthesis in P. aeruginosa. LaPhzS and LaPhzM, together
with LaPhzNO1, are the decorating enzymes that covert the dicarboxylic PDC to the
antibiotic myxin, whereas PhzS and PhzM are the decorating enzymes that convert the
monocarboxylic PCA to the virulence factor pyocyanin.17ꢀ20 PhzM catalyzes the
Nꢀmethylation step in pyocyanin biosynthesis, producing the intermediate N5ꢀmethylꢀPCA.
However, the Nꢀmethylated product has not been isolated. This is in contrast to the reactions
catalyzed by LaPhzM, which generated a variety of methylated products as described above.
One reason could be due to the unstable nature of N5ꢀmethylꢀPCA, as suggested previously.18
On the other hand, there could be other possibilities. For example, PCA might not be the
direct substrate of PhzM; instead, 1ꢀhydroxyphenazine (10), the product of PCA through a
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decarboxylative hydroxylation catalyzed by PhzS,17 could be the substrate of PhzM. This
hypothesis is in agreement with the previous observation that PhzM’s function required the
presence of PhzS in the reaction.18 To test if LaPhzM had any Nꢀmethylation activity, we
tested if the enzymes from strain OH13 were able to convert PCA to pyocyanin. When
LaPhzS was incubated with PCA in the presence of FAD and NADH, a small peak at 17.4
min was produced from PCA, which ran at 17.8 min on HPLC (Figure 5). This product gave
a m/z of 197.05 for [M + H]+, which is consistent with the mass of 1ꢀhydroxyphenazine (10)
(Figure S4E). When both LaPhzS and LaPhzM were incubated with PCA in the presence of
FAD, NADH and SAM, another small peak at 17.1 min was produced, while the peak at 17.4
min disappeared on HPLC (Figure 5). This product gave a m/z of 211.03 for [M + H]+ (Figure
S4F), which is coincident with the mass of pyocyanin. However, none of the two small peaks
coꢀmigrated with standard pyocyanin on HPLC, which had a retention time of 5.9 min
although it also gave a m/z of 211.09 for [M + H]+ (Figure 5 and Figure S4G). Therefore, the
product at 17.4 min is 1ꢀhydroxyphenazine (10), and the product at 17.1 min is
1ꢀmethoxyphenazine (11). The results show that, like PhzS, LaPhzS is able to catalyze the
decarboxylative hydroxylation of PCA to produce 1ꢀhydroxyphenazine, but LaPhzM can only
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convert
1ꢀhydroxyphenazine
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Nꢀmethyltransferase when PCA is the substrate. Thus, LaPhzM is an Oꢀmethyltransferase,
whereas PhzM is an Nꢀmethyltransferase. Finally, it should be pointed out that only a small
fraction of PCA was converted to products 10 and 11 by LaPhzS and LaPhzM, suggesting
that the phenazine monocarboxylic acid is not an optimal substrate for the Lysobacter
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Overview of the threeꢀdimensional structure of LaPhzM. In order to shine light on
the structural basis of the enzyme activity and substrate selectivity of LaPhzM, we
determined a crystallographic structure of LaPhzM in complex with the cofactor at 1.42 Å,
using the mitomycin 7ꢀOꢀmethyltransferase MmcR from Streptomyces lavendulae as an
initial search model for phasing.23 An overview of the LaPhzM structure is shown in Figure
S6. Similar to many other Oꢀmethyltransferases, LaPhzM forms a dimeric structure with a
dimerization domain in the Nꢀterminus and a catalytic domain with the α/β Rossmann fold in
the Cꢀterminus of each monomer. The overall fold of LaPhzM mirrors that of MmcR despite
the low sequence identity between the two proteins (Figure S7A).23 All the secondary
structural elements are also conserved in PhzM (Figure S7B) with a larger structural variation
in comparison to that of MmcR.
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The active site. The electron density around the cofactor binding site clearly indicates
an SꢀadenosylꢀLꢀhomocysteine (SAH), instead of a SAM, in the LaPhzM crystal structure
(Figure 6A). Since SAM was incubated with LaPhzM during crystallization, this observation
indicates that LaPhzM is capable of catalyzing the demethylation of SAM in the absence of
the phenazine substrate. The surrounding environment at the cofactor binding site of LaPhzM
closely resembles that of MmcR and other similar SAMꢀdependent Oꢀmethyltransferases
(Figure S8).23ꢀ24 The proteinaceous environment at the active site of LaPhzM possesses the
structural characteristics for the general acid/baseꢀmediated transmethylation of Class I
Oꢀmethyltransferases.24 A 3D structural alignment shows that the His251ꢀGlu306 pair takes
similar geometric arrangement as their corresponding residues (His259 and Glu313,
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respectively) in MmcR (Figure 6B), which have been suggested to form a hydrogen bond
network and be involved in deprotonating the hydroxyl group of the substrate.23 Interestingly,
Asp252 (Asp260 in MmcR) adjacent to His251 and SAH is also within the hydrogen bonding
distance with the substrate near the active site. Sequence alignments indicate that His251,
Asp252 and Glu306 are highly conserved in the LaPhzM homologs and other similar
Oꢀmethyltransferases. It remains to be tested whether Asp252 plays a crucial role in
positioning the substrate or/and proton shuttling during the catalytic process.24
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Modeling of the LaPhzMꢀsubstrate complex. Although the LaPhzM structure
determined in this study is in the substrateꢀfree form, cavity analysis by CAVER and
structural comparison with the MmcRꢀSAHꢀmitomycin A complex (PDB ID: 3GXO) suggest
that the characterized LaPhzMꢀSAH complex is in an open state for substrate binding.23, 25
Indeed, 1ꢀhydroxyꢀ6ꢀmethoxyphenazine (6), the intermediate methylated product from DHP,
can be fit well into the substrate binding pocket of LaPhzM by ligand docking (Figure 7). In
this model, the substrate entry is surrounded by helices α9, α10 and α15 in one monomer and
is capped by helix α'1 from the second monomer, which leaves a narrow channel for substrate
entry (Figures 7A and 7B). At the substrate binding site, 6 is surrounded by eleven residues in
a largely hydrophobic environment (Figure 7C). Particularly, two aromatic residues Tyr158
and Phe290 located in helices α10 and α15, respectively, are at the surface of LaPhzM facing
the solvent environment, presumably serving as the gate of the substrate entry. These two
residues are conserved only in the close homologs of LaPhzM with high sequence identity
and likely play an important role in defining the substrate preference of LaPhzM. Tyr158 and
Phe290 are absent in MmcR, which leaves a wider opening gate to accommodate the large
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polar tail of mitomysin A. Apart from the hydrophobic and van der Waals interactions, and
the hydrogen bond network involving the three residues mentioned above at the active site, a
few polar residues including Gln103, His107, Gln155 and His297 may also form hydrogen
bonds with the 1ꢀhydroxyl group, N5 and N10, respectively, in DHP and thus prepare the
substrate to the optimal orientation for transmethylation (Figure 7).
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Overall, the local environment at the substrate binding pocket of LaPhzM strongly
suggests that proximity may also be an important mechanism for determining the enzymatic
activity and phenazine selectivity as observed in the enzymatic activity assay. The higher
activity of LaPhzM to 1,6ꢀdihydroxyphenazine (DHP) in comparison to 1ꢀhydroxyphenazine
is likely due to the hydrogen bonding interactions between one hydroxy group of DHP and
Glu103/His107 that facilitate the orientation of the second hydroxy optimal for methyl
transfer (Figure 7). Among the three phenazine substrates tested, LaPhzM shows the lowest
activity to 6ꢀhydroxyꢀ1ꢀmethoxyphenazine N5ꢀoxide (2) (Figure S5). The proton in the
6ꢀhydroxyl group of 2 has been proposed to form a stable hydrogen bond with oxidized N5 in
a pseudo sixꢀmember ring (Figure S9).12 The presence of an intramolecular hydrogen bond
network in 2 may reduce the efficiency of deprotonation on the 6ꢀhydroxyl group mediated
by a hydrogen bonding with His251 and thus reduce the transmethylation rate.
Structural comparison of the substrate binding pocket in LaPhzM and PhzM. The
absence of the cofactor and substrate in the reported PhzM structure makes it difficult for a
direct comparison of the substrate binding pocket between the two structures. Nonetheless,
SAH can be modelled into the active site of PhzM using its homolog IOMT (PDB ID: 1FP2)
as a model, as demonstrated previously owing to the high structural similarity in this region.18
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When overlaying the two structures around the SAH binding site, the most noticeable
difference is the solvent accessibility at the substrate binding pocket. The helices α9, α10 and
α15 in PhzM are further away from the active site (up to 7 Å relative to the corresponding Cα
atoms in LaPhzM). In addition, different from LaPhzM, helix α'1 in PhzM does not cover the
opening of the active site. These differences in the local structural arrangement result in a
much more solvent exposed active site in PhzM (Figure 8), which is not optimal for SAM
and/or substrate binding and the reaction of transmethylation. These observations may
explain the difficulties in coꢀcrystalizing PhzM with a SAM/SAH.18
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As discussed above in pyocyanin biosynthesis, it was proposed that failing to detect the
product when incubating PhzM with SAM and the substrate PCA in the absence of PhzS was
probably due to the instability of the product N5ꢀmethylꢀPCA.18 However, no SAM
consumption was reported either in the study, while this would be expected if the
transmethylation did occur. The results from the structural comparison between PhzM and
LaPhzM provide structural insights in supporting that the alternative explanation may exist,
since a free PhzM as in the reported structure is not optimal for catalyzing the
transmethylation reaction. The interaction of PhzM with PhzS may be required for
desolvation of the active site, which is necessary for the stable cofactor/substrate binding and
efficient transmethylation. By interacting with PhzM, PhzS could either cap the substrate
binding pocket directly by itself, or trigger conformational changes in PhzM and lead to a
similar structural arrangement at the substrate binding pocket as observed in LaPhzM.
In conclusion, we have characterized the LaPhzM gene from strain OH13 and
demonstrated that LaPhzM is a SAMꢀdependent Oꢀmethyltransferase responsible for all
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methoxy formation in phenazine natural products isolated from strain OH13, including the
wellꢀknown antibiotic myxin. For the first time, we demonstrated the biosynthesis of myxin
from PDC, one of the two common precursors for all phenanzine natural products, through in
vitro reconstitution of the ringꢀdecorating steps using purified enzymes. Finally, we
determined the cofactorꢀbound Xꢀray crystal structure of LaPhzM at 1.42 Å resolution, which
provides molecular insights into the catalytic activity and Oꢀmethyl selectivity of the enzyme.
These findings will facilitate future efforts to improve myxin production in strain OH13
through metabolic pathway engineering, and the identification of the phenazineꢀdecorating
enzymes will find uses in chemoenzymatic syntheses of new phenazine antibiotics.
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310 ■ METHODS
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Cloning and Expression of LaPhzM in E. coli. LaPhzM was amplified from the
genomic DNA of strain OH13 (CGMCC No.7561), using primers LaPhzMꢀPF/LaPhzMꢀPR
(Table S1). The product was cloned into the EcoRI/HindIII sites in plasmid pET28a
(Novagen) and confirmed by DNA sequencing. This construct was introduced into E. coli
BL21(DE3) for expression. LaPhzM was purified using a NiꢀNTA column (GE Life Science)
and a size exclusion column (Sephadex 200) (Figure S2). A typical stock solution contained
LaPhzM of 30 mg/ml, in 50 mM Tris buffer pH 8, 200 mM NaCl and 1 mM DTT and was
stored at ꢀ80 °C. In the protein preparation for LaPhzM cyrstalization, the thrombin cleavge
site was removed from the LaPhzM expression construct.
Enzyme activity assays. Compound 2 was prepared from strain OH13 culture.16 PDC
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and PCA were purchased from Alligator Reagent Inc., China, and pyocyanin from
SigmaꢀAldrich. DHP (7) was purified by HPLC from the reaction mixture of PDC and
LaPhzS.16 The LaPhzS reaction was conducted in a 100 ꢁl mixture containing LaPhzS (1
ꢁM), the substrate (PDC or PCA, 100 ꢁM), NADH (100 ꢁM), and FAD (100 ꢁM) in
phosphate buffer (KH2PO4/K2HPO4, 100 mM, pH 7.0). The reaction mixture was incubated
at room temperature for 30 min. The reaction was quenched with an equal volume of ethyl
acetate, and the organic phase containing the product was collected and evaporated to dryness.
The residues were dissolved in methanol (100 ꢁl), and the supernatant was obtained by
centrifugation at 13,200 × g for 10 min. A fraction (20 ꢁl) of the supernatant was subjected to
reverseꢀphase HPLC analysis or LCꢀMS as described previously.16 The LaPhzNO1ꢀcatalyzed
Nꢀoxidation was carried out in the same conditions as that for LaPhzS, except NADH
replaced by NADPH (100 ꢁM). The LaPhzMꢀcatalyzed methyltransfer reaction was
conducted in a 100 ꢁl mixture containing LaPhzM (5 ꢁM), substrate (50 ꢁM), SAM (180
ꢁM), in Tris buffer (50 mM, pH 7.8). The mixture was incubated at room temperature for 30
min, and the reaction was stopped by adding ethyl acetate. To quantify the selectivity, kinetics
analysis was performed with compound 2 and SAM. Initial velocity of the reaction was
measured at a variety of substrate concentrations. The reaction was quantified by measuring
the SAM product, SAH, using reverseꢀphase HPLC (254 nm). A calibration curve of SAH
was prepared prior to the enzyme assays. These data were globally fitted to
MichaelisꢀMenten equation, yielding KM values. In the reactions to compare various potential
substrates for LaPhzM, the relative conversion rate was calculated based on the conversion
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rate of compound 2 to compound 4, which was set as 100%. The relative rate of other
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substrates converted by LaPhzM was expressed as the percent of that of compound 2
converted to compound 4.
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In reactions with PCA as substrate to test the activity of LaPhzS and LaPhzM in
pyocyanin biosynthesis in vitro, the procedures were the same as above, but with the
following modifications for HPLC analysis. A sample (20 ꢁl) was injected to a
reversedꢀphase column (Cosmosil, 4.6 ID × 250 mm) in an Agilent HPLC (1220 Infinity LC).
Water/0.05% formic acid (solvent A) and methanol 0.05% formic acid (solvent B) were used
as the mobile phases with a flow rate of 1.0 ml/min. The HPLC program was as follows:
5−25% B in the first 5 min, 25ꢀ80% B in 5ꢀ25 min, 100% B at 26ꢀ35 min, back to 5% B at 36
min, and maintained to 40 min. The phenazines were detected at 254 nm on a UV detector.
LCQꢀMS 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
1.5:1 before concentrating the protein to 20 mg/ml. LaPhzM was crystallized by sittingꢀdrop
vapor diffusion at room temperature (~ 295 K). The protein solution at 20 mg/ml was mixed
in 1:1 volume ratio with the reservoir solution containing 0.2 M ammonium formate, 18 ꢀ 22 %
w/v PEG 3350. Crystals typically reached the maximum size in about one week. Santovac® 5
Cryo Oil (Hampton Research) was used as the cryoprotectant when harvesting the crystal.
Xꢀray Diffraction Data Collection, Processing and Refinement. Xꢀray diffraction
data were collected on Beamline 9ꢀ2 at the Stanford Synchrotron Radiation Lightsource
(SSRL), with a 6M Pixel Array Detector. A set of 1200 diffraction images was collected at
12,658 eV with an oscillation angle of 0.15°. Diffraction data were integrated with the XDS
program package,26 and subsequently processed using programs of the CCP4 suite.27 The
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phase was determined by molecular replacement using structure of the mitomycin
7ꢀOꢀmethyltransferase MmcR from Streptomyces lavendulae (PDB code 3gwz, 34% of
sequence identity)23 as the search model and employing the BALBES automated molecular
replacement pipeline server.28 Further refinement and structural validation was carried out by
using REFMAC5, COOT and MolProbity.29ꢀ31 The diffraction data and refinement statistics
are listed in Tables S2 and S3, respectively. All but the 6xHis tag and the first 9 residues in
the Nꢀterminus of the native protein have been included in the model. The programs COOT
and PyMOL (The PyMOL Molecular Graphics System, Version 1.7.1 Schrödinger) were used
for structural analysis and for generating figures. The atomic coordinates and experimental
data (code 6C5B) have been deposited in the Protein Data Bank (www.wwpdb.org).
Molecular Docking. The prediction of LaPhzM–substrate binding was performed with
Autodock Vina employing default parameters using a grid with dimensions of 26ꢂ×ꢂ20ꢂ×ꢂ20 Å
centered at the substrate binding cavity.32 Water molecules were removed from the LaPhzM
structure in preparation of docking. The coordinates for methylated phenazines were
modified from the structures downloaded from the PubChem database. The Gasteiger charges
and hydrogens were added to the receptor structure and phenazines with AutoDocktools.33
The receptor structure was treated as a rigid body during docking analysis to exclude the
uncertainty caused by the structural flexibilities.
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385 ■ ASSOCIATED CONTENT
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Supporting Information
Details of primer sequences, statistics for data processing and refinement, multiple
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amino acid sequence alignment, SDSꢀPAGE of proteins, enzyme kinetics, protein secondary
structural elements, models of cofactor and substrate binding environments in protein
structures, and spectroscopic data for identification of phenazines. This material is available
free of charge via the Internet at http://pubs.acs.org.
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393 ■ AUTHOR INFORMATION
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Corresponding Author:
*Eꢀmail: ldu3@unl.edu; Telephone: +402ꢀ4722998. Fax: +402ꢀ4729402
*Eꢀmail: limei.zhang@unl.edu; Telephone: +402ꢀ4722967. Fax: +402ꢀ4727842
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Author Contributions:
L.D. and L.Z. conceived and designed experiments. J.J. and D.B. carried out experiments.
L.D., L.Z., J.J and A.S. analyzed data. J.J., L.D. and L.Z. wrote the manuscript.
402 ■ ACKNOWLEDGMENTS
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This work was supported in part by the NIH (R01AI097260), UNL Redox Biology
Center, a Faculty Seed Grant and a Revision Award from UNL. We thank Y. Zhao for helpful
discussion at the beginning of this project, R. Cerny and K. Wulser for technical assistance,
and H. Li for the insightful discussion on the charge distribution for molecular docking.
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Thomashow, L. S. (2010) Diversity and evolution of the phenazine biosynthesis
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[6] Selezska, K., Kazmierczak, M., Musken, M., Garbe, J., Schobert, M., Haussler, S.,
Wiehlmann, L., Rohde, C., and Sikorski, J. (2012) Pseudomonas aeruginosa
population structure revisited under environmental focus: impact of water quality and
phage pressure. Environ. Microbiol. 14, 1952ꢀ1967.
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Figure 1. Chemical structure of selected phenazine natural products isolated from
Pseudomonas aeruginosa and Lysobacter antibioticus. Some of the functional groups on the
phenazine ring are color highlighted to indicate the decorations relevant to this research.
Figure 2. HPLC analysis of the reaction product of LaPhzM with
6ꢀhydroxyꢀ1ꢀmethoxyphenazine N5ꢀoxide (2) as substrate. (A) LaPhzM + substrate with
SAM. (B) Boiled LaPhzM + substrate with SAM. (C) Substrate 2.
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Figure 3. HPLC analysis of reaction products of LaPhzM with 1,6ꢀdihydroxyphenazine
(DHP, 7) as substrate. From top to bottom, the HPLC traces are for reactions incubated for 0,
15, 45, 90 min, respectively.
Figure 4. In vitro reconstitution of myxin biosynthesis. In addition to myxin and other known
phenazines, the reactions produced a new compound (9), a phenazine with dimethoxy and
diꢀNꢀoxide, which had not been isolated from strain OH13.
Figure 5. HPLC analysis of reaction products of LaPhzS and LaPhzM with PCA as substrate.
Pyocyanin was also included as a reference.
Figure 6. The active site of the LaPhzM. (A) The 2FoꢀFc electron density map around the
cofactor binding site. The map is illustrated in light blue color and contoured at 3σ. The
cofactor product SꢀadenosylꢀLꢀhomocysteine (SAH) is represented by sticks. (B) The stick
representation of the active site in LaPhzM (cyan carbon atoms) overlaid with the
MmcRꢀSAHꢀmitomycin A (MMA) complex (PDB ID: 3GXO, gray carbon atoms). The
residues in LaPhzM are labeled in cyan fonts and the corresponding ones in MmcR are
labeled in black fonts in the round bracket. The nitrogen atoms are in blue, oxygen in red and
sulfur in yellow.
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Figure 7. The substrate binding pocket of LaPhzM with 6 fit into the structure by
autodocking. (A) and (B) are the surface representations of LaPhzM viewed from the
proteinꢀsolvent interface toward the substrate binding pocket with and without the second
monomer of the dimer, respectively. The black arrow indicates the proposed narrow substrate
entry tunnel between helices α9, α10 and α15 from one monomer and helix α'1 from the
second monomer. The surface presentation is colored by the underlying atoms with nitrogen
in blue and oxygen in red. The surface around carbon atoms is colored white for one monoer,
and yellow for the second promoter for clarity. The local environment at the substrate binding
pocket is shown in (C). The carbon atoms are colored cyan for the amino acid residues and
gray for SAH and 6. The water molecule is shown in the red sphere. SAH, 6 and the amino
acid residues within 4 Å of 6 are represented by sticks.
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Figure 8. Comparison of the substrate binding pocket of PhzM (PDB ID: 2IP2) and LaPhzM.
(A) A ribbon presentation of PhzM (blue) overlaid with LaPhzM (light gray). Only the
residues from AA121 to the end of the Cꢀterminus are included for clarity. (B) A surface
presentation around the active site of PhzM viewed from the proteinꢀsolvent interface. The
surface presentation is colored by the underlying atoms with nitrogen in blue and oxygen in
red. The surface around carbon atoms is colored white for one monomer and yellow for the
second promoter for clarity. SAH was modelled to the active site of PhzM guided by its
homolog IOMT (PDB ID: 1FP2); 5ꢀMeꢀPCA (N5ꢀmethylphenazine carboxylic acid) was
manually placed near to SAH for the purpose of demonstration.
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Figure 1.
4
5
6
7
8
COO
COOH
COOH
N
O
10
9
1
4
10a
N
N
9a
N
N
N
2
3
8
7
4a
5a
N
H2N
N
R
N
N
6
5
9
CH3
COOH
CH3
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Phenazine
1-carboxylic
acid (PCA)
Phenazine
1,6-dicarboxylic
acid (PDC)
Aeruginosin A (R=H)
Aeruginosin B (R=SO3H)
Phenazine
Pyocyanin
O
OH
O
OCH3
O
OH
OCH3 OH
O
OH
N+
N+
N+
N
N
N+
N+
O
N
N+
O
N
N
N
OH
OCH3
OCH3
OCH3
OCH3
OCH3
6
5
1 (Myxin)
3 (Iodinin)
2
4
OH
O
OH
OH
OCH3
O
OCH3
N+
N
N
N
N
N+
N
N
N+
O
N
OH
OH
OCH3
7 (DHP)
8 (DHPO)
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557
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559
560
561
562
Figure 1. Chemical structure of selected phenazine natural products isolated from
Pseudomonas aeruginosa and Lysobacter antibioticus. Some of the functional groups on the
phenazine ring are color highlighted to indicate the decorations relevant to this research.
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Figure 2.
O-
OH
O-
OCH3
N+
N+
LaPhzM
SAM
N
N
OCH3
OCH3
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Figure 2. HPLC analysis of the reaction product of LaPhzM with
6ꢀhydroxyꢀ1ꢀmethoxyphenazine N5ꢀoxide (2) as substrate. (A) LaPhzM + substrate with
SAM. (B) Boiled LaPhzM + substrate with SAM. (C) Substrate 2.
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Figure 3.
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583
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Figure 3. HPLC analysis of reaction products of LaPhzM with 1,6ꢀdihydroxyphenazine
(DHP, 7) as substrate. From top to bottom, the HPLC traces are for reactions incubated for 0,
15, 45, 90 min, respectively.
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