Functional Analysis of Cytochrome P450s Involved in Streptovaricin

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Functional Analysis of Cytochrome P450s Involved in Streptovaricin Biosynthesis and Generation of Anti-MRSA Analogues Yuanzhen Liu, Xu Chen, Zhengyuan Li, Wei Xu, Weixin Tao, Jie Wu, Jian Yang, Zixin Deng, and Yuhui Sun ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00467 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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ACS Chemical Biology

Functional Analysis of Cytochrome P450s Involved in Streptovaricin Biosynthesis and Generation of Anti-MRSA Analogues Yuanzhen Liu1, Xu Chen1, Zhengyuan Li1, Wei Xu1, Weixin Tao1, Jie Wu2, Jian Yang2, Zixin Deng1,3 and Yuhui Sun1*

1

Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan

University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, People’s Republic of China 2

Renmin Hospital of Wuhan University, Wuhan 430060, People’s Republic of

China 3

State Key Laboratory of Microbial Metabolism, School of Life Sciences &

Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

* To whom correspondence should be addressed. E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT The streptovaricins, chemically related to the rifamycins, are highly effective antibacterial

agents

particularly

against

mycobacteria.

Herein,

a

bioassay-guided investigation of Streptomyces spectabilis CCTCC M2017417 has led to the characterization of streptovaricins as potent compounds against methicillin-resistant Staphylococcus aureus (MRSA). We identified the streptovaricin biosynthetic gene cluster from S. spectabilis CCTCC M2017417 based on genomic sequencing and bioinformatic analysis. Targeted in-frame deletion of five cytochrome P450 genes (stvP1-P5) resulted in the identification of four new streptovaricin analogues, and revealed the functions of these genes as follows: stvP1, stvP4 and stvP5 are responsible for the hydroxylation of C-20, Me-24 and C-28, respectively; stvP2 is possibly involved in formation of the methylenedioxy bridge, and stvP3, a conserved gene found in the biosynthetic cluster for naphthalenic ansamycins, might be related to the formation of naphthalene ring. Biochemical verification of the hydroxylase activity of StvP1, StvP4 and StvP5 was performed, and StvP1 showed unexpected biocatalytic specificity and promiscuity. More importantly, anti-MRSA studies of streptovaricins and derivatives revealed significant structure-activity relationships (SARs): the hydroxyl group at C-28 plays a vital role in antibacterial activity; the hydroxyl group at C-20 substantially enhances activity in the absence of the methoxycarbonyl side chain at C-24, which can increase the activity regardless of the presence of a hydroxyl group at C-20; the inner lactone ring between C-21 and C-24 shows a positive effect on activity. This work provides meaningful information on the SARs of streptovaricins,

and

demonstrates

the

utility

of


the

engineering

of

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streptovaricins to yield novel anti-MRSA molecules.

INTRODUCTION Antibiotic resistance poses a serious threat to public health, causing many difficult-to-treat infections in humans.1 In particular, the methicillin-resistant Staphylococcus aureus (MRSA) is now considered as a "superbug" that has resulted in major illness, death and huge economic losses, due to its virulence and wide distribution in community and hospital settings.2, 3 Therefore, there is an urgent need to develop new agents to target these pathogens. Natural products have proved to be an important reservoir for discovering anti-MRSA antibiotics such as vancomycin and daptomycin.4, 5 Engineered biosynthesis can complement this conventional time-consuming discovery process and represents an attractive approach to creating molecular diversity for drug discovery. Novel “unnatural” natural products can be obtained through direct genetic modification of the biosynthetic pathway in the producing strains or by combinatorial biosynthesis in a heterologous host.6, 7 The naphthalenic ansamycin streptovaricins, chemically related to the rifamycins, were first discovered from Streptomyces spectabilis NRRL 2494 in 1957, and the chemical structures of streptovaricin A and C were determined in 1968 (Figure 1).8, 9 Afterwards, a series of analogues, including streptovaricin F, G, J, K10 and U,11 the possible biosynthetic intermediates damavaricin C and D,12 and the trace components protostreptovaricins I-V,13 were successfully separated and identified from S. spectabilis spp. (Figure 1). Most recently, five new streptovaricins named as ansavaricins A-E, were isolated from Streptomyces sp. S012.14 They have a broad antibacterial spectrum against both Gram-positive and Gram-negative bacteria, especially Mycobacterium



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tuberculosis.15, 16 The antibacterial activity of streptovaricins results from its inhibition of DNA-dependent RNA synthesis in prokaryotes. The degree of bacterial

inhibition

is

different

among

individual

members

of

the

streptovaricins.17 However, the structure-activity relationships (SARs) of streptovaricins are still obscure. In terms of structural variations, these 23-membered macrocyclic lactams differ from other ansamycin antibiotics mainly in the different extent of oxidation of the ansa bridge, e.g. C-20 and C-28 hydroxylation, oxidation of Me-24, and dehydrogenation of C-21. Especially, C-6 and C-11 are linked via a methylenedioxy bridge (MDB) to form the characteristic naphthodioxine moiety.10 Structurally, streptovaricins are members of the ansamycin family of macrolactam natural products. In general, the biosynthesis of ansamycins requires a Type I modular polyketide synthase (PKS),18 which initiates the polyketide chain with 3-amino-5-hydroxybenzoic acid (AHBA),19-22 followed by multiple steps of chain elongation with various extender units on subsequent modules of the PKS.23, 24 The extended chain is then cyclized and released from the PKS by an amide synthase, and undergoes further post-PKS modifications.25-27 However, to the best of our knowledge, the post-PKS tailoring steps of streptovaricins including hydroxylation,

methylation,

acetylation

and

redox

reactions

(on

the

naphthalene ring and ansa bridge), remain to be elucidated. Streptovaricin biosynthesis has previously only been studied by feeding experiments with isotope-labeled

putative

[carboxy-14C]AHBA and

precursors.19,

28

[methyl-14C]methionine

The

incorporation

demonstrated

that

of the

aromatic chromophore of streptovaricins originates from AHBA, and the methyl group at C-3 of the naphthalenic chromophore, the methylenedioxy, and


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methoxy carbons are derived from methionine.19, 28 In this study, we report the analysis of the biosynthetic gene cluster of streptovaricins and of their anti-MRSA activities for the first time. To understand the anti-MRSA SARs of streptovaricins and generate more potent streptovaricin analogues, experiments were undertaken to delineate the role of each post-PKS tailoring enzymes, especially focusing on the cytochrome P450 enzymes, to pave the way for further structure and activity optimization.

RESULTS AND DISCUSSION Isolation and Identification of Streptovaricins and Protostreptovaricins from S. spectabilis CCTCC M2017417. During our screening for anti-MRSA antibiotics, a strain designated S. spectabilis CCTCC M2017417 was isolated from a soil sample collected in the campus of Wuhan University in China. Its 16S ribosomal RNA (rRNA) gene sequence (accession number KY604743) showed 99.4% similarity to S. spectabilis NBRC13424T (accession number AB184393) and the ethyl acetate extract was found to be active against S. aureus ATCC 29213 and S. aureus ATCC 43300 (methicillin-resistant). Subsequently, a bioassay-guided fractionation of the anti-MRSA metabolites from strain S. spectabilis CCTCC M2017417 led to the identification of streptovaricins and protostreptovaricins (1-6) (Figure 1, Figure S1). The molecular formula of compound 1, the most abundant metabolite, was determined to be C40H51NO14 by ESI-HRMS, which is the same with that of streptovaricin C. The 1H and

13

C NMR data (Table S1), together with the 2D

NMR spectra (1H-1H COSY, HSQC, and HMBC (Figure S2)), allowed the full characterization of 1 as streptovaricin C (Figure 1).29 Similarly, the structure of



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5 were established as the same with that of protostreptovaricin I, based on ESI-HRMS and NMR data (Figure S1, Figure S3).13 The chemical structures of 2–4 and 6 are inferred unambiguously based on the ESI-HRMS analysis (Figure S1). Although only six streptovaricins were identified from the strain CCTCC M2017417, other structural analogues, such as streptovaricin A, B, E, J, K and U, were also detected in the crude extracts of this strain. These findings indicate that S. spectabilis CCTCC M2017417 possesses a great potential to produce a wide range of streptovaricin analogues with potentially novel structures and activities via biosynthetic engineering. Genomic Sequencing and In silico Analysis of the Streptovaricin Biosynthetic Gene Cluster. To identify the streptovaricin biosynthetic gene cluster, whole genome shotgun sequencing of S. spectabilis CCTCC M2017417 was performed. With the help of antiSMASH analysis,30 two putative ansamycin biosynthetic gene clusters were found in the 9.7 Mb genome with 72.6% G+C content. One of them contains PKSs with eight modules and shows high homology with the reported biosynthetic gene cluster for the benzenic ansamycin herbimycin, and the other gene cluster, which we have denoted as stv, has eleven Type I PKS modules that exactly correspond with assembly of the streptovaricin backbone. Besides five genes encoding Type I modular PKSs (stvA-E), another valuable clue is that the cluster consists of a set of AHBA biosynthesis genes (stvG-N) and an amide synthase (stvF), which would be necessary for streptovaricin biosynthesis. The putative stv cluster (accession number KY593296) spans ~95 kb of genomic DNA, and 41 open reading frames (ORFs) with predicted functions that were assigned based on homology analysis (Figure 2; Table S2). In addition, five cytochrome


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P450 genes (stvP1-P5), four oxidoreductase-encoding genes (stvO1-O4), three

methyltransferase-encoding

genes

(stvM1-M3),

two

acyltransferase-encoding genes (stvA1-A2) and two regulatory genes (stvR1-R2) were also included in the stv cluster. Interestingly, there are as many as five genes (stvP1-P5) in the stv cluster that encode proteins with homology to the cytochrome P450 family. stvP1 encodes a protein sharing homology with Rif-Orf16, a cytochrome P450 which related to the conversion of rifamycin SV to rifamycin B.25 However, StvP2 showed no homology to any reported ansamycin post-PKS tailoring enzyme. StvP3 is a conserved protein in naphthalenic ansamycin biosynthetic pathways, and is highly homologous to Rif-Orf0 (identity 76%) in rifamycins biosynthesis.17, 18 Nat-Orf0 (identity 69%) in naphthomycins biosynthesis31 and Cxm4 (identity 79%) in chaxamycins biosynthesis.27 StvP4 and StvP5 are homologous to Rif-Orf13, a cytochrome P450 monooxygenase with no obvious role in rifamycins biosynthesis.25 Besides, in the four oxidoreductase-encoding genes (stvO1-O4), stvO4 encodes a protein highly homologous to Rif-Orf19, the 3-(3-hydroxyphenyl)propionate hydroxylase-like protein that catalyze the formation of the naphthalene ring of rifamycins.17,

32

Of the two

acyltransferase-encoding genes (stvA1& A2), stvA2 encodes a protein with homology to Rif-Orf20, an O-acetyltransferase that catalyzes the O acetylation of

C-25

during

rifamycin

biosynthesis.25

Among

the

three

methyltransferase-encoding genes (stvM1-M3), stvM2 encodes a protein homologous to Cxm24, which is speculated to be responsible for C-methylation of the C-3 of AHBA in the naphthalene ring.27 Based on bioinformatic analysis and our current understanding of



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ansamycin natural products, a possible streptovaricin biosynthetic pathway is proposed (Figure 3). Initiated with the AHBA starter unit, the polyketide backbone of streptovaricins is extended with the incorporation of one malonyl-CoA and two methylmalonyl-CoA extender units to generate a tetraketide tethered to the acyl carrier protein domain (ACP) of module 3. The discrete

four-module

StvA

and

two-module

StvB

may

provide

an

inter-molecule space allowing the accessibility and the oxidation of the tetraketide intermediate, followed by a spontaneous intra-molecular cyclization to form the naphthalene ring. The tetraketide with naphthalene ring is then extended with one malonyl-CoA and six methylmalonyl-CoA extender units, producing a linear polyketide chain tethered to the ACP of module 10. Subsequently, the nascent polyketide chain is transferred, hydrolyzed, and cyclized by the amide synthase StvF to form the 23-membered macrocyclic intermediate, prostreptovaricin. Next, it is aromatized by two parallel pathways, including the dehydration and the dehydrogenation steps between C-7 and C-8, and further tailoring steps, to generate both protostreptovaricins and streptovaricins. Genetic and Structural Evidence Reveals that StvP1, StvP4 and StvP5 Are Responsible for the Hydroxylation of Streptovaricins at C-20, Me-24 and C-28, Respectively. To understand their roles in streptovaricins formation, all the five P450 genes were individually knocked out by targeted in-frame deletion (Figure S4-S8, Table S3-S5). The HPLC analysis showed that 3-fold higher production of streptovaricin C (1) was accumulated in stvP1 disrupted mutant ∆stvP1, while the production of streptovaricin G (3) was totally abolished (Figure 4). Due to the fact that 3 is the C-20 hydroxylated product of


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1, StvP1 can be functionally assigned as the C-20 hydroxylase in streptovaricin biosynthesis. Complementation of the ∆stvP1 mutant with stvP1 under the control of the PermE* promoter restored the production of streptovaricin G nearly to wild-type levels (Figure S9). For the mutant ∆stvP4, the production of streptovaricin C (1) was terminated, but the yields of streptovaricin D (2) increased nearly 100-fold higher in ∆stvP4 (Figure 4). Considering the fact that 1 is actually the C-28 hydroxylated products of 2, we can infer that StvP4 functions as the C-28 hydroxylase of streptovaricins. In the ∆stvP5 mutant, the production of streptovaricin C, D, G and F (1-4) were all abolished, in favor of a series of new metabolites (Figure 4). Complementation of the ∆stvP4 and ∆stvP5 with stvP4 and stvP5, respectively, under the control of the PermE* promoter restored the production of streptovaricins nearly to wild-type levels (Figure S9). We isolated 7 to 10 as yellow oils from a scaled-up liquid culture of ∆stvP4 and ∆stvP5 for structural characterization (Figure 4, Figure S10). Compounds 7 and 8 were isolated from a 5 liter liquid culture of mutant ∆stvP4. The molecular formulae of 7 and 8 were assigned as C39H47NO13 (calcd. [M+H]+ 738.3120) and C38H49NO12 (calcd. [M+H]+ 712.3328) based on the [M+H]+ ion peak at m/z 738.3101 and 712.3309 in the LC-ESI-HRMS spectrum, respectively (Figure S11). Further extensive spectroscopic analysis, including 1D and 2D NMR, and comparison with literature data, allowed the elucidating of the structures of 7 and 8 to be 28-dehydroxyl-streptovaricin F and 6-O-methyl-28-dehydroxyl-damavaricin C, respectively. 7 is readily recognized by the fact that Me-28 (δH 0.70 (3H, d, J=6.7 Hz)) in 7 attached to the aliphatic carbon (δC 37.1) but not an oxygenated carbon at C-28 in streptovaricin F (Figure S12). Interestingly, 8



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features two methoxyl groups, and the location of 6-OCH3 was ascertained by clear HMBC correlation from 6-OMe (δH 3.81, δC 61.8) to C-6 (δC 161.7) (Figure S13). Thus, the structures of 7 and 8 were determined as shown in Figure 4. The titers of 7 and 8 were determined to be ~10 mg/L and ~6 mg/L respectively. Obviously, identification of 7 and 8 further indicated that StvP4 is the hydroxylase that responsible for hydroxylation of C-28 of streptovaricins. Compounds 9 and 10 were purified from mutant ∆stvP5. LC-ESI-HRMS spectra of 9 and 10 showed [M+H]+ ion peaks at m/z 742.3436 and 726.3486, and their molecular formulae were determined to be C39H51NO13 (calcd. [M+H]+ 742.3433) and C39H51NO12 (calcd. [M+H]+ 726.3484), respectively (Figure S11). Since the molecular weights of 9 and 10 are 44 amu smaller than that of streptovaricin G (3) and streptovaricin C (1), we speculated that the methoxycarbonyl side-chain (-COOMe) attached to C-24 is replaced by a methyl group (-Me) in 9 and 10. The

13

C NMR spectrum of 9 showed 39

carbons, one carbon less than 3. A comparison of the NMR spectra of 9 and 3 revealed that a methoxyl group was missing in 9, while a new aliphatic methyl proton signal (δH 1.00 (3H, d, J=6.8Hz)) was found. The only methoxyl group found in 3 lies in the side-chain attached to C-24. Therefore, 9 is likely to be the 24-methylated derivative of 3. This was supported by the 1H-1H COSY correlation of Me-24/H-14, and HMBC correlations of Me-24 at δH 1.00 to C-24 (δC 32.7), C-23 (δC 80.1) and C-25 (δC 70.7) (Figure S14). Based on similar reasoning (Figure S15), the structure of 10 was determined to be the 24-methylated derivative of 1. Accordingly, the structures of 9 and 10 were established as shown in Figure 4. The titers of 9 and 10 were determined to be ~7 mg/L and ~4 mg/L, respectively. All the carbon and proton signals of 7 to 10


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were assigned unambiguously (Table S6) based on the 1D and 2D NMR (Figure S12-S16), and comparison with related known compounds. Taken together, characterization of 9 and 10 revealed that cytochrome P450 monooxgenase StvP5 is involved in the formation of methoxylcarbonyl side chain of streptovaricins and may responsible for a single oxidation (from alkane to alcohol) or multi-step oxidation (from alkane to carboxylic acid) of Me-24. Actually, it is fairly common that some P450s can catalyze sequential oxidation of methyl groups to carboxylic acids, especially in steroid metabolism.33 Functional analysis of StvP3 and StvP2. StvP3 is fairly conserved and homologs are found in all reported naphthalenic ansamycin biosynthetic clusters, but not in any known benzenic ansamycin biosynthetic clusters, which may indicate that this enzyme is related to the formation of the naphthalene ring.17 Inactivation of stvP3 abolished production of all known streptovaricins and related metabolites (Figure 4), while the complementation of the ∆stvP3 with stvP3 under the control of the PermE* promoter restored the production of streptovaricins to wild-type levels (Figure S9), which indicated that StvP3 may act at an early stage in streptovaricin biosynthesis. This result is also in agreement with the proposal that the naphthalene ring formation could occur during polyketide backbone elongation. StvP2 encodes a protein that shares 61% homology with a cytochrome P450 hydroxylase (locus tag: SCATT_p09400) from S. cattleya DSM 46488. To test its function, we first in-frame deleted the stvP2 gene in S. spectabilis CCTCC M2017417. The resultant ∆stvP2 only produced protostreptovaricin I (5), which bears no MDB moiety but has a hydroxyl group at C-6 (Figure 1,



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Figure 4). Complementation of the ∆stvP2 with stvP2 under the control of the PermE* promoter restored the production of streptovaricins to wild-type levels (Figure S9). MDB formation is one of the key steps in streptovaricin biosynthesis. It has been reported that MDB is catalyzed by cytochrome P450s in higher plants.34-37 We thus initially hypothesized that a cytochrome P450 could also be responsible for the MDB formation in streptovaricin biosynthesis. Among the five identified cytochrome P450s in the stv cluster, StvP1, StvP4 and StvP5 are functionally assigned as the hydroxylases of C-20, Me-24 and C-28, respectively. Apart from these, StvP3 is unlikely to participate in the MDB formation based on the above bioinformatic and genetic inactivation results. This left the possibility of StvP2 being the catalyst for MDB formation, however, there was no reaction between StvP2 and 5. According to previous reports, several plant P450s have been characterized to catalyze the formation of MDB moieties between a methoxy group and a nearby hydroxyl group. Thus, compound 8 bearing a methoxy group at C-6 in the vicinity of C-11 was an attractive candidate substrate for MDB formation. We therefore used 8 isolated from ∆stvP4 to test the in vitro activity of StvP2, but there was no reaction. Alternatively, 8 was not a substrate for StvP2, one possible reason for this could be the extremely strict substrate recognition of MDB-forming enzymes.36 In vitro Assays Reveal the Catalytic Specificity of StvP1, StvP4 and StvP5. To further validate the function of StvP1, StvP4 and StvP5, the corresponding PCR-amplified fragments containing each entire gene were cloned into pET28a(+) vector and overexpressed in E. coli BL21(DE3). The N-terminal His6-tagged StvP1, StvP4 and StvP5 were purified to near homogeneity (Figure S17) and reacted with potential substrates. For StvP1, its


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ability to hydroxylate at C-20 of the predicted substrates, i.e., compounds 1, 2, 5, 8 and 10, using a typical heterologous spinach ferredoxin and ferredoxin reductase system38 was tested. Reaction mixtures were quenched at different times by the addition of ethyl acetate and analyzed by both HPLC and LC-ESI-HRMS. As expected, 1 and 10 were converted to the corresponding C-20 hydroxylated products 3 and 9, respectively. The time-course analysis of this hydroxylation was performed as shown in Figure 5, showing the expected increase with reaction time and confirming that StvP1 is competent as the C-20 hydroxylase in streptovaricin biosynthesis. However, StvP1 showed poor activity towards compounds 2, 5 and 8 even reacted for overnight (128

32

>128

32

4

32

1

ATCC 25904

a

0.125

128

0.125

0.125

>128

64

>128

16

8

32

1

ATCC 43300b

0.25

64

0.5

1

>128

16

>128

16

2

16

1

0.25

64

1

1

>128

64

>128

16

8

32

1

0.25

64

0.5

1

>128

128

>128

16

8

32

1

ATCC 29213

USA300 LAC

b

b

USA400 MW2 a

Methicillin-sensitive Staphylococcus aureus (MSSA) strains.

b

Methicillin-resistant Staphylococcus aureus (MRSA) strains.


(Control)

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TOC 90x90mm (300 x 300 DPI)


Functional Analysis of Cytochrome P450s Involved in Streptovaricin

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