Jun Xu, Taifo Mahmud* and Heinz G. Floss*

Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195

Identification and Characterization of 27-O-Demethylrifamycin SV Methyltransferase

Background

Rifamycin B, one of the prominent members of the ansamitocin family, is a microbial secondary metabolite antibiotic produced by Amicolatopsis mediterranei. Its synthetically modified derivatives are clinically used in the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Recently, rifamycin derivatives have also shown excellent activity against HIV and oncogenic viruses. However, many pathogen bacteria develop resistance to rifamycins with high frequency.The ansa chain of Rifamycin B has been proposed to be synthesized from acetate and propionate units catalyzed by a large multifunctional complex enzyme, type I polyketide synthases (PKSs). The starter molecule for this polyketide assembly is derived from 3-amino-5-hydroxybenzoic acid (AHBA). Although great progress has been made in the last few years on the understanding of the biosyntheses of AHBA and the ansa chain of rifamycin B, little is known regarding the post-PKS modification genes and the tailoring processes leading to rifamycin B. Here we report the identification and characterization of the Orf14, an S-adenosyl-L-methionine dependent methyltransferase that is involved in post-PKS modification of rifamycin B biosynthesis.

Cloning and Inactivation of orf 14

Isolation and Structure Elucidation of Accumulated Compound

III. Post-PKS Modification Genes

II. AHBA Biosynthesis

I. PKSL 1 2 3 4 5 6 7 8 9 10

BamH I

rifA rifB rifC rifD rifE rifF

rifG rifHrifI

rifK rifLrifM

rifNorf1

orf0

H2N

OH

S-E

2 kb

1.5 kb

rifOrifJ

rifP rifQorf2 orf3

orf4 orf5 orf6 orf7orf8

orf9orf10

orf11orf17

orf18 orf19orf20orf12

orf13orf14

orf15 orf16

OH O

CH3

O

CH3 CH3

OH

CH3

OH

CH3

OH

CH3

OH

CH3 CH3

O

Orf14 was subcloned into Bluescript II KS-, and frame shift inactivation was done at XhoI site. Double homologous recombination mutant of Amycolatopsis mediterranei S699 (mutant MT1401XH) was obtained.

13 1614 15

xho I xho I xho I xho I1.6 Kb 4.9 Kb1.5 Kb

12

pBluescript II KS-

Oxidation of 27-O-Demethylrifamycin SV to 27-O-Demethylsifamycin SCatalyzed by Divalent Ions

Genetic Organization of the Rifamycin Biosynthetic Gene Cluster

From fermentation broth of mutant MT1401XH, 27-O-demethylrifamycin SV was isolated as a major metabolite, together with traces of 25-O-deacetyl-27-O-demethylrifamycin SV, 27-O-demethylrifamycin S, and rifamycin W. Their chemical structures were elucidated based on mass spectroscopy and NMR data.

Over Expression of orf 14

In order to obtain His6-fusion protein, two primers were designed and PCR was carried out to amplify the orf14. The PCR product was inserted into pRSET B, a T7 promoter expression vector, and the product was transferred into E. coli BL21 by heat-pulse transformation. SDS polyacrylamide gel electrophoresis confirmed that the right size protein was over-expressed. The protein was purified by Ni-NTA spin column.

Characterization of Orf14

D. Substrate Specificity

E. Kinetic parameters

Conclusion Proposed biosynthetic pathway to Rifamycin B.

27-O-Demethylrifamycin SV

27-O-Demethylrifamycin S

Cu2+

Mn2+

Ca2+, Co2+, Fe2+, Mg2+, Ni2+, Zn2+

Acknowledgements

This work was supported by a research grant AI 20264 from the National Institutes of Health. Acquisition of the Bruker Esquire ion trap mass spectrometer was supported by the National Science Foundation through grant No. 9807748.

C. Effects of Divalent ions

635 640 645 650 655 660 665 m/z0

1

2

3

4

5

6

5x10Intens. 6: MS, Time=0-0.35min (#1-#65)

654.8

641.0

656.2

670.6668.6657.6

680.0 682.5 685.0 687.5 690.0 692.5 695.0 697.5 700.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens. 4: MS, Time=0-0.4min (#1-#76)

696.4

682.4

683.2

684.3

697.1

698.3

699.2

680.0 682.5 685.0 687.5 690.0 692.5 695.0 697.5 700.0 m/z0

1

2

3

4x10Intens. 18: MS, Time=0-0.75min (#1-#133)

680.3

681.3

694.3682.3

683.3 690.3684.3 686.3 693.3

696.3

695.3697.2 698.4

1

42

ESI-MS data for enzyme assays with different rifamycins as substrate: 1. 27-O-Demethylrifamycin SV;2. 27-O-Demethylrifamycin S; 3. Rifamycin W; 4. 25-O-Deacyl-27-O-demethylrifamycin SV

27-O-Demethylrifamycin SV appears to be the right substrate for the methyltransferase, Orf14 (Figure 1)

QuickTime™ and aPlanar RGB decompressor

are needed to see this picture.

38 KD

Vmax=142.8 nM/sec

Km=19.3 uM

635 640 645 650 655 660 665 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

6x10Intens. 8: MS, Time=0-0.23min (#1-#45)

640.7

3Rif SV

A. Optimal Buffer B. Optimal pH Value

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

H3CCOO

CH3H3CO

OCH2COOH

HOHO

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3HO

CH3HO

O

OHO

H3C

H3CNH

O

O CH3

CH3CH3

OHOH

CH3HO

CH3HO

O

OOHCH2OH

H3C

H3C

HO

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

H3CCOO

CH3HO

OH

HOHOH3C

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

H3CCOO

CH3H3CO

OH

HOHOH3C

Methylation

Acylation

Rifamycin W 25-O-Deacetyl-27-O-demethyl rifamycin SV

27-O-Demethylrifamycin SV

Rifamycin SV Rifamycin B

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

H3CCOO

CH3H3CO

OH

HOHOH3C

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

HO

CH3HO

OH

HOHOH3C

Rifamycin SV27-O-Demethylrifamycin SV 25-O-Deacetyl-27-O-demethyl rifamycin SV

NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

H3 CCOO

CH3HO

OH

HOHOH3C NH

O

CH3O

O

O CH3

CH3CH3

OHOH

CH3

H3 CCOO

CH3HO

O

OHOH3C

27-O-Demethylrifamycin S

NH

O

O CH3

CH3CH3

OHOH

CH3

HO

CH3HO

O

OOHCH2OH

H3C

H3C

HO

Rifamycin W