Lantibiotic Biosynthesis: Identifying and studying

1
Lantibiotic Biosynthesis Identifying and
studying the cyclase enzymes involved in nisin
and subtillin production
Okeley, N. M., Paul, M., Stasser, J. P.,
Blackburn, N. and van der Donk, W. A.
Biochemistry (2003) ASAP 10/30/03
2
Lantibiotics

• defense peptides
• peptide derived antimicrobial
• agents
• ribosomally synthesized
• post-translationally modified
• by a multienzyme complex
• proteolytically activated
• genes for biosynthesis are
• organised in clusters
• 19-38 amino acids long
• Produced by, and highly active
• against, Gram positive bacteria

3
Pink Bugs and Purple Bugs

• 1884, Hans Christian Gram - A Dane working in
  Berlin
• Studying lung tissue from patients who had died
  of pneumonia
• () violet colour and (-) pink
• The Gram negative bacteria are low in
  peptidiglycan so do not take
• up the crystal violet stain.

4
How to make Lantibiotics

• After ribosomal synthesis of prepeptide
• (contains a leader sequence), modification
  occurs based mainly
• on just three amino acids
• Serine
• Theonine
• Cysteine
• The two key reactions are
• the selective dehydration of Ser and Tyr residues
  resulting in the
• ?,?-unsat amino acids didehydroalanine and
  didehydrobutyrine and
• (2) Intramolecular nucleophillic addition

5
(No Transcript)
6
Pore Formation Type A Lantibiotics

• Ramseier (1960)
• Reisinger (1980)
• Roberts (1996)
• Wormhole model

in vivo formation of transmembrane pores.
1. Peptides become amphiphilic upon binding to
membranes. 2. This enables insertion into the
outer membrane. (Hydrophobic) 3. Electrical
potential of membrane drives the peptide residues
through carrying surface phospholipid along.
7
Binding to Lipid II Type B Lantibiotics

• Binds not to D-Ala-D-Ala but to the
  disaccharide-PP moiety of Lipid II.
• a new target binding site not used by any
  current antibacterial drug

Sahl, H-G and Bierbaum, G. Annu. Rev. Microbiol.,
1998, 52 41-79
8
Nisin (Type I)

• pentacyclic peptide, posttransalationally
  mofified lantibiotic
• possessing high antimicrobial activity against
  Gram Bacteria
• used in food preservation for over 30 years
  (discovered 1940s)
• 11 genes involved in nisin synthesis
  nisABTCIPRKFEG

• separate dehydratase and cyclase (LanB and LanC)
  for Nisin
• ( subtilin) production.

Runar, R., Beerthuyzen, de Vos, W. M., Saris, P.
E. J. and Kuipers, O. P. Microbiology, (1999)
145, 1227-1233, Effects of gene disruptions in
the nisin gene cluster of Lactococcus Lactis on
nisin production and producer immunity
9
SpaC and NisC, the Cyclases involved in Subtilin
and Nisin Biosynthesis, are Zinc Proteins.
Okeley, N. M., Paul, M., Stasser, J. P.,
Blackburn, N. and van der Donk, W. A.
Biochemistry (2003) ASAP 10/30/03 University of
illinois at Urbana-Chamapign
In an effort to gain further insight into the
mechanism by which lantibiotics are
biosynthesized, the cyclase enzymes involved in
the synthesis of nisin and subtilin have been
cloned, expressed and purified .. and studied
by ICP, EXAFS and for cyclase activity.
10
Over expression and purification of proteins
SpaC 49,353 Da

• Allows production of His-Tag Fusion proteins

• Both proteins are produced at 25 ?C with IPTG
  induction (ampicillin).
• Both are soluble
• SpaC (NcoI/BamHI) purified by anion-exchange and
  gel filtration c/tography
• nisC (XhoI/BamHI) purified by nickel c/tography
  followed by thrombin cleavage
• and size exclusion c/tography

11
Metal Content
ICP

• ICP for nisC 0.61 Zns per protein and 0.64
  for SpaC

and PAR Assay

• 4-(2-pyridylazo)resorcinol assay
• denature with DTNB
• 5,5-dithiobis(2-nitrobenzoic acid)

• RT in 20 mM Tris buffer _at_ pH 7.4
• Abs _at_ 500 nm

12
EXAFS

• Stanford beamline
• Frozen glasses in 30 ethylene glycol
• k 15 Å-1
• max R value 2.3 Å
• values consistent with Zn-thiol coordination

• Best Fit 2Zn-S 2Zn-N/O
• Histidine values elevated
• Model Zn(Cys)2(His)2

it seems likely that Zn is coordinated by two
cysteine residues, but the identity of the
accompanying O/N ligands is less certain
13
Mutagenesis

• C349A and C303A

• GST fusion protein using pGEX6p1 vector

EXAFS (C349A) Best Fit now- 1Zn-S 2Zn-N 1Zn-O
C349 is a ligand for Zn
14
Putative role of Zinc (simply structural?)

• Activate Cysteine thiol of peptide toward
  nucleophilic attack
• of the dha or dhb residues in the same
  peptide(Intramolecular addition)

• Many S-alkylation proteins
• contain Zn
• net negative charge at Zn site-
• increased thiolate reactivity
• lower C pKa from 8.5 to 6.5
• nisin ring formation without
• enzyme only occurs at pH gt 8

Demonstrate cyclase activity in vitro ?
15
Peptide Synthesis

• Built SpaC truncated substrate 2-32 (actual has
  57 residues) Yield 0.3!
• Incubated with SpaC
• HPLC monitoring showed new cyclic product but..
• rate was identical to control! Reasons??

16
References
Runar, R., Beerthuyzen, de Vos, W. M., Saris, P.
E. J. and Kuipers, O. P. Microbiology, (1999)
145, p1227-1233 Okeley, N. M., Paul, M.,
Stasser, J. P., Blackburn, N. and van der Donk,
W. A. Biochemistry, (2003) ASAP 10/30/03 Sahl,
H-G and Bierbaum, G. Annu. Rev. Microbiol.,
(1998) 52, p41-79. Pag U, Sahl HG. Curr Pharm
Des., (2002) 8, p815-33. http//www.uni-tuebinge
n.de/jung/lanti.html