The biosynthesis of albicidin

(1)

NRPS-1

NRPS-2

NRPS-3

NRPS-4

NRPS-5

The biosynthesis of albicidin

Daniel Petras

1

**_{*, Andi Mainz}**

1

_{, Benjamin Hempel}

1

_{, Stéphane Cociancich}

2

_{, Monique Royer}

2

_{, Roderich D. Süssmuth}

1

_{Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany.}

2

_{Cirad, UMR BGPI, F-34398 Montpellier Cedex5, France.}

INTRODUCTION

RESULTS AND DISCUSSION

REFERENCES

CONTACT INFORMATION

Albicidin is a potent inhibitor of bacterial DNA gyrase with IC

₅₀

values in a nM range, produced by the sugarcane pathogenic bacterium

Xanthomonas albilineans [1]. The structure of albicidin remained unclear for more than three decades after its first description by Birch et al.

[2]. After the identification and sequencing of three gene islands, responsible for the albicidin biosynthesis, a PKS-NRPS hybrid, build up by

three enzymes, Alb01, Alb05 and Alb09 was proposed for the albicidin assembly [3, 4]. Most recently we were able to solve the hitherto

unknown structure, revealing a unique polyaromatic oligopeptide mainly composed of p-amino benzoic acids [5, 6]. In-vitro studies of the

non-ribosomal albicidin assembly line provided further insights into the biosynthetic machinery of albicidin. Together with our bioinformatic

investigations we were able to propose a comprehensive biochemical assembly, expanding the non-ribosomal code of adenylation domains

with p-amino benzoic acid derivatives. Furthermore our study reveals a new type of dehydratase domain responsible for the in situ formation

and incorporation of cyano-alanine [6].

Figure 3. In silico substrate selectivity of adenylation domains in albicidin NRPS modules. a) Selectivity-conferring residues of A domains

are derived from sequence alignments to GrsA. Note that the highly conserved residue D235 (red), which commonly interacts with the α- amino group of the substrate, is preserved only for the Asn/Cya-activating NRPS-2* A domain.

However, a new aspartic acid appears for all A domains that have been shown to activate p-aminobenzoic acid derivatives (highlighted in red). Relevant interactions in the substrate binding pockets of A domains are shown for (b) the Phe-activating GrsA (PDB 1amu)11 and (c) the structural model of NRPS-1 (based on homology modeling with the ITASSER webtool using GrsA as template).

1. Hashimi, S.M., Wall, M.K., Smith, A.B., Maxwell, A. & Birch, R.G.

Antimicrobial agents and chemotherapy 51, 181-7 (2007).

2. Birch R. G., P.S.S. Phytopathology 73, 1368-1374 (1983).

3. Huang, G., Zhang, L., Birch, R.G., Microbiology 147 (Pt 3),

631-42 ( 2001).

4. Royer, M., Costet, L., Vivien, E., Bes, M., Cousin, A., Damais, A.,

Pieretti, I., Savin, A., Megessier, S,, Viard, M., Frutos, R., Gabriel,

D.W., Rott, P.C., Mol Plant Microbe Interact. 17, 414-27, (2004)

5. Süssmuth, R.D., Kretz, J., Schubert, V., Pesic, D., Hügelland, M.,

Royer, M., Cociancich, S., Rott, P., Kerwat, D., Grätz, S., ALBICIDIN

DERIVATIVES,

THEIR

USE AND

SYNTHESIS,

Patent,

WO2014125075, (2014)

6. Cociancich, S., Pesic, D., Petras, D., Uhlmann, S., Kretz, J.,

Schubert, V., Vieweg, L., Duplan, S., Marguerettaz, M., Noell, J.,

Pieretti, I., Hügelland, M., Kemper, S., Mainz, A., Rott, P., Royer, M.,

Süssmuth, R.D. Nat. Chem. Biol., 2015, published online

* daniel.petras@chem.tu-berlin.de

MCA

pABA

Cya

pABA pMBA

pMBA

Figure 1. The structure of the albicidin. Albicidin is composed of a

methylated derivative of p-coumaric acid (MCA), the non-proteinogenic α -amino acid cyanoalanine (Cya) as well as the aromatic δ-amino acids p-aminobenzoic acid (pABA, pABA) and 4-amino-2-hydroxy- 3-methoxybenzoic acid (pMBA, pMBA) [5, 6].

0 10 20 30 40 50 60 70 80 90 100 110 NRPS-1 Blank pAB A pMB A _pABA-2,3-diOH L-Phe L-Asn 0 10 20 30 40 50 60 70 80 90 100 110 NRPS-3 Blank pAB A pMB A pAB A-2,3-diOH L-Phe L-Asn 0 10 20 30 40 50 60 70 80 90 100 110 NRPS-2* Blank pAB A pMB A L-Asn D-Asn L-Asp L-Cy a D-Cy a 0 10 20 30 40 50 60 70 80 90 100 110 NRPS-2 Blank pAB A pMB A

L-Asn D-Asn L-Asp

L-Cy a D-Cy a 0 10 20 30 40 50 60 70 80 90 100 110 NRPS-4 Blank pAB A pAB A-2-OH pAB A-3-OH pMB A pAB A-2,3-diOH 0 10 20 30 40 50 60 70 80 90 100 110 NRPS-5 Blank pAB A pAB A-2-OH pAB A-3-OH pMB A pAB A-2,3-diOH

Figure 5. Substrate specificities of NRPS activation domains in albicidin biosynthesis. Relative turnover of activated substrates are shown. Radioactive

ATP/PPi exchange assays were performed for the A domains of NRPS-1, NRPS-2, NRPS-2*, NRPS-3, NRPS-4 and NRPS-5. Normalization to 100% refers to 65.9 µCi/mol (NRPS-1), 1160.8 µCi/mol (NRPS2 and NRPS-2*), 168.3 µCi/mol (NRPS-3), 259.8 µCi/mol (NRPS-4) and 557,2 µCi/mol (NRPS-5), respectively. Experiments were performed in duplicates and error bars of standard deviation are shown.

precursor synthesis PKS

NRPS

tailoring proteins resistance genes NRPS associated proteins

esterase

transposase genes/ regulation albicidin transporter

,

Figure 2. Gene cluster of albicidin biosynthesis. The gene machinery

responsible for albicidin biosynthesis is located on three loci (XALB1-3). XALB1 contains the main genes. XALB2 and XALB3 encode each only one ORF, a phosphopantetheinyl transferase and a heat shock protein HtpG [3, 4].

a

b

c

(pABA)

(pHBA-CoA) (pABA) (pAHBA) (pAHBA)

(Asn)

AL

AT

A

(Asn)

A

C

A

C

A

TE

C

?

A

KS

KR

KS

ACP DH ACP ACP T0 _T1 _T2 T3 T4

T2*

_T2*

T2*

T5 MT PKS-1 PKS-2 PKS-3 NRPS-1 NRPS-2 NRPS-2* NRPS-3 NRPS-4 NRPS-5

X

Alb08 pMBA Alb20 Alb18/ pabC pABA pAHBA pHBA-CoA Chorismate Alb17/ pabAB Alb02 SAM SAH Gln Glu Alb12 NADP O , NADPH₂ + NADH NAD+ pyruvate Alb07 CoA, ATP AMP, PP i pyruvate Alb09 ATP PP_i OH O OH O O OH OH O OH OH O S CoA NH2 O OH O O OH NH2 O OH NH2 O OH OH NH2 O S OH NH₂ O S OM e NH₂ O S OM e OH

Alb09

Alb04

Alb13

Al

b01

S O HN O HO M eO HN O NH N O NH O OH HN O HN O HO M eO HO M eO HN O NH N O NH O OH O S S O HN O NH N O NH O OH S O NH N O NH O OH S O NH O OH S O OH OH S O S O OH O O N H₂ NH₂ OH S

-+ ATP

- PPi

O O N H₂ NH₂ AMP B O O N H NH₂ S P O O -O -O O N H₂ NH₂ S O NH₂ S O N N H₂ S H N H B O

ATP

AMP

PO

4

3-NO HIT

Module A domain signature Predicted substrate

NRPS-1 GrsA

A V K Y V A N

D

A K

D

A W T I A A I C K

NO HIT

NRPS-3

A V K Y V A N

D

A K

NRPS-2*

D

L T K I G E V G K

E L T Y V H A - - R

Asx

NRPS-2 NRPS-4

A I K Y F S I

D

M K

NO HIT

NRPS-5

A I K Y F S I

D

M K

NO HIT

235 236 239 278 299 301 322 330 331 517 D235 D321 K221 Y260 A217 T278 W239 K517 O O K510 NH3 I330 NH3 O O HO N H O O NH3 H2N H3C A322 CH3 OH H3 2 N O O N313 NH O K517 D235 I330 C331 A236 W239 A322 I299 A301 T278 K510 A217 D321 V218 K221 N313 V287 A289 Y260

Figure 4. Model of albicidin biosynthesis. a) Proposed biosynthetic assembly line for albicidin. Substrates of the NRPS are indicated at the A domains. PKS and

NRPS modules are color-coded in blue and green, respectively. b) Suggested pathways for the biosynthesis of the MCA-1 precursor pHBA-CoA as well as the δ- amino acids pABA and pMBA. c) Suggested mechanism for the transformation of activated Asn into Cya by Alb04. Conventional activation of Asn by adenylation is mediated by the A domain of Alb04. The substrate is subsequently stored as a thioester at the 4’-phosphopantetheine arm (T2* domain). A second activation occurs by phosphorylation of the Asn side-chain, which finally leads to a formal elimination of water and thus to the formation of Cya.