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Glycosyltransferases and more, important tools for drug design A. Bechthold, Universität Freiburg. Compounds with anticancer activity. Compounds with antimicrobial activity. 9 Sugars 6 GTs. 6 Sugars 4 GTs. 17 Sugars 10 GTs. UrdGT1a. SaqGT5. SaqGT6. SaqGT1. SaqGT2. SaqGT3. UrdGT2. - PowerPoint PPT Presentation
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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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