Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function

The polyketide synthase CTB1 is demonstrated to catalyze pyrone formation thereby expanding the known biosynthetic repertoire of thioesterase domains in iterative, non-reducing polyketide synthases

Influence of Lag Effect, Soil Release, And Climate Change on Watershed Anthropogenic Nitrogen Inputs and Riverine Export Dynamics

This study demonstrates the importance of the nitrogen-leaching lag effect, soil nitrogen release, and climate change on anthropogenic N inputs (NANI) and riverine total nitrogen (TN) export dynamics using a 30-yr record for the Yongan River watershed in eastern China. Cross-correlation analysis indicated a 7-yr, 5-yr, and 4-yr lag time in riverine TN export in response to changes in NANI, temperature, and drained agricultural land area, respectively. Enhanced by warmer temperature and improved agricultural drainage, the upper 20 cm of agricultural soils released 270 kg N ha(-1) between 1980 and 2009. Climate change also increased the fractional export of NANI to river. An empirical model (R(2) = 0.96) for annual riverine TN flux incorporating these influencing factors estimated 35%, 41%, and 24% of riverine TN flux originated from the soil N pool, NANI, and background N sources, respectively. The model forecasted an increase of 45%, 25%, and 6% and a decrease of 13% in riverine TN flux from 2010 to 2030 under continued development, climate change, status-quo, and tackling scenarios, respectively. The lag effect, soil N release, and climate change delay riverine TN export reductions with respect to decreases in NANI and should be considered in developing and evaluating N management measures

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of <i>in vitro</i> “domain swapped” NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs <i>in vitro</i>, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis

Structural and Biochemical Analysis of Protein–Protein Interactions Between the Acyl‐Carrier Protein and Product Template Domain

In fungal non-reducing polyketide synthases (NR-PKS) the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism-based crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-of-function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides

Paradigm Shift for Radical S-Adenosyl- l -methionine Reactions: The Organometallic Intermediate ω Is Central to Catalysis

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.ISSN:0002-7863ISSN:1520-512