Functional characterization of TtnD and TtnF, unveiling new insights into tautomycetin biosynthesis.
The biosynthetic gene cluster for tautomycetin (TTN), a highly potent and selective protein phosphatase (PP) inhibitor isolated from Streptomyces griseochromogenes, has recently been cloned and sequenced. To better understand the transformations responsible for converting the post-polyketide synthase product into the exciting anticancer and immunosuppressive chemotherapeutic candidate TTN, we produced and characterized new analogues resulting from inactivation of two genes, ttnD and ttnF, in S. griseochromogenes. Inactivation of ttnD and ttnF, which encode for putative decarboxylase and dehydratase enzymes, respectively, afforded mutant strains SB13013 and SB13014. The DeltattnD mutant SB13013 accumulated four new TTN analogues, TTN D-1, TTN D-2, TTN D-3, and TTN D-4, whereas the DeltattnF mutant accumulated only one new TTN analogue, TTN F-1. The accumulation of these new TTN analogues defines the function of TtnD and TtnF and the timing of their chemistries in relation to installation of the C5 ketone moiety within TTN. Notably, all new analogues possess a structurally distinguishing carboxylic acid moiety, revealing that TtnD apparently cannot catalyze decarboxylation in the absence of TtnF. Additionally, cytotoxicity and PP inhibition assays reveal the importance of the functional groups installed by TtnDF and, consistent with earlier proposals, the C2''-C5 fragment of TTN to be a critical structural determinant behind the important and unique PP-1 selectivity displayed by TTN.
Luo
Yinggang
Y
Division of Pharmaceutical Sciences, University of Wisconsin, USA.
Li
Wenli
W
Ju
Jianhua
J
Yuan
Qiuping
Q
Peters
Noel R
NR
Hoffmann
F Michael
FM
Huang
Sheng-Xiong
SX
Bugni
Tim S
TS
Rajski
Scott
S
Osada
Hiroyuki
H
Shen
Ben
B
D000475:Alkenes / Q000737:chemistry; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005663:Furans / Q000494:pharmacology; D020868:Gene Silencing; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008055:Lipids / Q000096:biosynthesis* / Q000494:pharmacology; D005810:Multigene Family; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
D000475:Alkenes; D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005663:Furans; D008055:Lipids; C058744:tautomycetin; D010749:Phosphoprotein Phosphatases; D002262:Carboxy-Lyases; D006836:Hydro-Lyases