Structure and Catalytic Mechanism of a Cyclic Dipeptide Prenyltransferase with Broad Substrate Promiscuity

• Published in: Journal of molecular biology 2012-09, Vol.422 (1), p.87-99
• Main Authors: Schuller, Jan Michael, Zocher, Georg, Liebhold, Mike, Xie, Xiulan, Stahl, Mark, Li, Shu-Ming, Stehle, Thilo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aspergillus fumigatus - enzymology; Catalysis; crystal structure; Crystallography, X-Ray; diketopiperazines; Dimethylallyltranstransferase - chemistry; Dimethylallyltranstransferase - metabolism; dipeptides; Dipeptides - chemistry; Dipeptides - metabolism; enzyme mechanism; Fungal Proteins - chemistry; Fungal Proteins - metabolism; fungi; Hydrophobic and Hydrophilic Interactions; hydrophobicity; indole prenylation; Indoles - chemistry; Indoles - metabolism; Molecular Sequence Data; mutagenesis; nuclear magnetic resonance spectroscopy; Peptides, Cyclic - chemistry; Peptides, Cyclic - metabolism; prenyl transferase; Prenylation; protein structure; Structure-Activity Relationship; Substrate Specificity; Tryptophan - chemistry; Tryptophan - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2012.05.033

Fungal indole prenyltransferases (PTs) typically act on specific substrates, and they are able to prenylate their target compounds with remarkably high regio- and stereoselectivity. Similar to several indole PTs characterized to date, the cyclic dipeptide N-prenyltransferase (CdpNPT) is able to prenylate a range of diverse substrates, thus exhibiting an unusually broad substrate promiscuity. To define the structural basis for this promiscuity, we have determined crystal structures of unliganded CdpNPT and of a ternary complex of CdpNPT bound to (S)-benzodiazepinedione and thiolodiphosphate. Analysis of the structures reveals a limited number of specific interactions with (S)-benzodiazepinedione, which projects into a largely hydrophobic surface. This surface can also accommodate other substrates, explaining the ability of the enzyme to prenylate a range of compounds. The location of the bound substrates suggests a likely reaction mechanism for the conversion of (S)-benzodiazepinedione. Structure-guided mutagenesis experiments confirm that, in addition to (S)-benzodiazepinedione, CdpNPT can also act on (R)-benzodiazepinedione and several cyclic dipeptides, albeit with relaxed specificity. Finally, nuclear magnetic resonance spectroscopy demonstrates that CdpNPT is a C-3 reverse PT that catalyzes the formation of C-3β prenylated indolines from diketopiperazines of tryptophan-containing cyclic dipeptides. [Display omitted] ► Structure of the fungal prenyltransferase CdpNPT. ► Structural basis for substrate promiscuity in CdpNPT. ► Insight into catalysis of regioselective C-3β prenylation. ► Results help to rationalize and modify a prenylation reaction.