Glycosyltransferases and more, important tools for drug design

A. Bechthold, Universitt Freiburg

Compounds with anticancer activity

Compounds with antimicrobial activity

9 Sugars6 GTs6 Sugars4 GTs17 Sugars10 GTs

UrdGT2UrdGT1aUrdGT1cUrdGT1bLanGT2LanGT1LanGT4LanGT3LanGT1LanGT4SaqGT5SaqGT2SaqGT1SaqGT6SaqGT3 SaqGT4 (?)SaqGT3 SaqGT4 (?)

Can we use glycosyltransferases as tools to generate novel antibiotics?

LanGT2LanGT1LanGT1LanGT3LanGT4LanGT4

LanGT1LanGT3LanGT4 UrdGT2

More than 50 new compounds by combining landomycin- and urdamycin biosynthetic genes !Landomycin AUrdamycin A

How do glycosyltransferases look like?

UrdGT2MurGOleDOleIGtfAGtfBGtfD

UrdGT2

Can we combine N- and C-terminal parts of different glycosyltransferases?

+

LanGT1/LndGT1: 390 aa(25% non -identical aa)

LanGT1-like activity:

Landomycin with 5 and 6 sugarsLndGT1-like activity:

Landomycin with 2, 3 and 4 sugarsno activity:

Landomycin with 1 sugar

N-term.C-term.

LanGT1 likeactivity %LndGT1 likeactivity%No activity%385210

Which amino acids are responsible for substrate specificity?

Mutations affecting the acceptor substrate specificity of a glycosyltransferase

Hoffmeister et al., 2001, ChemBiolHoffmeister et al., 2002, ChemBiolHancock et al., 2006, Curr Opin Chem BiolWilliams et al., 2007, Nat Chem BiolWilliams et al., 2008, ChemBiol

NC4v1a: 1-3

v1b: region between 3 and 4

v2a: 4 v2b: 4-5

v3: 6-7

N-term.C-term.v1a v1b v2a v2b v3v1a (64 aa): 1-3v1b (19 aa): region between 3 and 4v2a (23 aa): 4 v2b (35 aa): 4-5v3 (68 aa): 6-7

C

LanGT1 likeactivity %LndGT1 likeactivity%No activity%195467915

NC4v1a: 1-3

v1b: region between 3 and 4

v2a: 4 v2b: 4-5

v3: 6-7

LanGT1 likeactivity %LndGT1 likeactivity%No activity%3466

NC4v1a: 1-3

v1b: region between 3 and 4

v2a: 4 v2b: 4-5

v3: 6-7

I64 V67 A68 V64 L67 A68 A73 E74 A75 L73 Q74 S75 D82 E82v1b

LanGT1 likeactivity %LndGT1 likeactivity%No activity%19542773

G107 R110 R107 G110L119 Y121 V122 F124 I119 F121 I122 L124T129 D129v2b

LanGT1 likeactivity %LndGT1 likeactivity%No activity%67915277217712213960

G107 R110 R107 G110L119 Y121 V122 F124 I119 F121 I122 L124T129 D129v2b

LanGT1 likeactivity %LndGT1 likeactivity%No activity%12772121782967432599

T89 S89R93 P94 D97 P93 Q94 E97E101 A101A104 D104v2a

LanGT1 likeactivity %LndGT1 likeactivity%No activity%771221927354223

I64 V67 A68 R93 P94 D97 L119 Y121 V122 F124 V64 L67 G68 P93 Q94 E97 I119 F121 I122 L124

LanGT1 likeactivity %LndGT1 likeactivity%No activity%

NC4

3 4 54ERecognitionPositioning

Glycosyltransferases and more, important tools for drug design

A. Bechthold, Universitt Freiburg

Weitnauer et al., ChemBiol,2001Weitnauer et al., Microbiol, 2002Mosbacher et al., J Mol Biol 2003Treede et al., Mol. Microbiol 2003Weitnauer et al., ChemBiol 2004Mosbacher et al., J Mol Biol, 2005Treede et al., Ap Env Mic, 2005Hofmann et al., ChemBiol, 2005Boll et al., JBC, 2006

Avilamycin AGavibamycin L1Activity against MRSA (MIC)42logP (pH: 7.4)3.32.9Protein binding(%)75.574.3Survival rate of mice(1.25 mg/kg)33%67%Successful co-crystallization with rRNA NoYes

AviE2AviQ1

Gavibamycin L1Gavibamycin N1

Activity against MRSA (MIC)216Activity against Streptococcus pyrogenes (MIC)28Activity against Enterococcus faecalis (MIC)18

AviX12

AviG6

Search for compounds acting like avilamycins

Development of a two-component assay using the FACS technology (in cooperation with N.Q, Le, Dr. I. Mecklenbruker, Prof. Dr. D. Bumann)

noavilamycinavilamycin6g/mlDetection of strains producing compounds acting like avilamycin A

A. Luzhetskyy, G. Weitnauer, H. Yu Thank you very much!!

GlycosyltrasferasesOxygenasesMembrane proteins (White biotechnology)RecombineeringChargeM. DaumU. HardterS. HermannGntherLinnenbrinkK. ProbstK. PetzkeJ. HrlePeintnerS. EbelingE. WelleC. KrauthH. J. SchnellM. FedoryshynT. StrobelM. FedoryshynA. SchandelmeierC. JilgH. WeiM. WeberS. Gaiss

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