The Ternary Complex of PrnB (the Second Enzyme in the Pyrrolnitrin Biosynthesis Pathway), Tryptophan, and Cyanide Yields New Mechanistic Insights into the Indolamine Dioxygenase Superfamily

Abstract

Pyrrolnitrin (3-chloro-4-(2-nitro-3-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-L-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both L-tryptophan or 7-Cl-L-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In L-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr 321 and Ser 332 play a key role in binding these groups. The structures suggest that catalysis requires an L-configured substrate. Isothermal titration calorimetry data suggest D-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications.Pyrrolnitrin is a broad-spectrum potent antifungal compound (1) first isolated from Pseudomonas pyromania (2) and the active component of PYRO-ACE (treatment for fungal infections of skin). The gene cluster responsible for pyrrolnitrin biosynthesis was identified in Pseudomonas fluorescens (BL915) (3, 4) and subsequently in Pseudomonas pyrrocinia, Burkholderia cepacia LT4-12-W, Myxococcus fulvus Mx f147, and other pyrrolnitrin producing bacteria (5). Four conserved enzymes are involved in pyrrolnitrin biosynthesis and named PrnA, PrnB, PrnC, and PrnD, reflecting their order in catalysis. Introduction of the entire cluster to Escherichia coli results in the production of pyrrolnitrin, thereby demonstrating that the four genes are sufficient and essential for pyrrolnitrin biosynthesis in vivo (3). Genetic manipulation in P. fluorescens BL915 has identified the intermediate products from each enzyme in the pyrrolnitrin pathway, leading to the current model for the biosynthetic pathway (4). The first enzyme, PrnA (tryptophan 7-halogenase), incorporates the chlorine into the substrate tryptophan (6). Structural and biochemical analyses of both tryptophan 7 and 5-halogenase (7-10) have established a novel chemical mechanism of hypohalous acid formation at the flavin cofactor, followed by ...

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications

“…37 Its structure was resolved for the cyanide complex and two ternary complexes with both l -tryptophan or 7-Cl- l -tryptophan and cyanide. 37,38 …”

Tryptophan oxygenation: mechanistic considerations

“…The indole of 7-chloro-L-tryptophan binds in the same manner as seen previously, but the main-chain amino and carboxy groups have rotated away from the iron to allow CN to bind to it. Notably the ternary complex resulted in ordering a loop adjacent to the active site [15]. Measurement of binding affinity suggested an obligate order of substrate binding, with CN binding first [15], which was also recently shown to be the case for TDO [16].…”

“…Notably the ternary complex resulted in ordering a loop adjacent to the active site [15]. Measurement of binding affinity suggested an obligate order of substrate binding, with CN binding first [15], which was also recently shown to be the case for TDO [16]. Unusually, when bound to PrnB, CN adopted a bent conformation (like that expected for oxygen), with the nitrogen pointing towards an oxyanion hole.…”

“…More recent studies on PrnB highlighted mimicry of the ternary complex with 7-chloro-L-tryptophan and cyanide (CN) bound [15]. The indole of 7-chloro-L-tryptophan binds in the same manner as seen previously, but the main-chain amino and carboxy groups have rotated away from the iron to allow CN to bind to it.…”

“…Despite repeated attempts, to date, the purified PrnB has not been turned over with its presumed substrate in vitro [15]. Activity has been detected in crude extracts only, and, for this reason, data from this system have and should be treated with caution.…”