Dihydrofolate reductase-like protein inactivates hemiaminal pharmacophore for self-resistance in safracin biosynthesis.

Dihydrofolate reductase (DHFR), a housekeeping enzyme in primary metabolism, has been extensively studied as a model of acid-base catalysis and a clinic drug target. Herein, we investigated the enzymology of a DHFR-like protein SacH in safracin (SAC) biosynthesis, which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance. Furthermore, based on the crystal structure of SacH-NADPH-SAC-A ternary complexes and mutagenesis, we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore. These findings expand the functions of DHFR family proteins, reveal that the common reaction can be catalyzed by distinct family of enzymes, and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.

Structure-Based Mechanistic Insights into ColB1, a Flavoprotein Functioning in-trans in the 2,2'-Bipyridine Assembly Line for Cysteine Dehydrogenation.

The recruitment of trans-acting enzymes by nonribosomal peptide synthetase (NRPS) assembly line is rarely reported. ColB1 is a flavin-dependent dehydrogenase that is recruited by an NRPS terminal condensation domain (Ct domain) and catalyzes peptidyl carrier protein (PCP)-tethered cysteine dehydrogenation in collismycin biosynthesis. We here report the crystal structure of ColB1 complexed with FAD and reveal a typical structural fold of acyl-CoA dehydrogenases (ACADs). However, ColB1 shows distinct structural features from ACADs in substrate recognition both at the entrance of and inside the active site. Site-directed mutagenesis and substrate modeling establish a Glu393-mediated catalytic mechanism, by which the cysteine substrate is sandwiched between Glu393 and FAD to facilitate Cα proton abstraction and Cß hydride migration. A ColB1-PCP-Ct complex model is generated, providing structural basis for the unique recruitment interactions between ColB1 and the associated NRPS. These results add insights into the mechanisms by which trans-acting enzymes function in an assembly line.

The Influence of Short-Term Heavy Rainfall on Hydraulic Characteristics and Rill Formation in the Yuanmou Dry-Hot Valley.

Rill erosion is one of the major environmental problems in the world; it is an important factor with regard to land degradation and has a serious impact on production and daily life in the region. The widely distributed Yuanmou group stratum promotes the development of rill erosion, whereby the strong time-concentrated rainfall and the alternating arid-humid climate prepare the ground for the development of rills in soils. Therefore, a study of the processes of slope rill erosion was carried out, and a gravel-soil slope in the Yuanmou dry-hot valley was chosen to simulate short-term heavy rainfall (25 mm/h) (No. 1 plot) and moderate rainfall (15 mm/h) (No. 2 plot), to study the erosion processes of soil and the dynamic characteristics of runoff involved in erosion. The study results showed that the width of runoff was significantly different between the two plots, while the depth of runoff was not significantly different. During the rill formation process, the width of the two plots first decreased and then increased with increasing washout duration, while its depth did not change significantly. Flow was the key factor in determining the hydraulic characteristics of runoff, and it had a significant or extremely significant positive correlation with hydraulic characteristics parameters, except in the case of Fr (Froude number) (r = 0.039). The total sediment content (CS) of plot No. 1 (0.158 g/cm3) was significantly different from that of plot No. 2 (0.153 g/cm3), and both CSs in the two plots decreased with increasing washout duration. The CS had an extremely significant negative correlation with τ (runoff shear force) (r = -0.863 **) and DW-f (Darcy-Weisbach drag coefficient) (r = -0.863 **) and a significant negative correlation with Re (Reynolds number) (r = -0.735 *) in the short-term heavy rainfall experiment, while the CS had a significant positive correlation with V (velocity) (r = 0.814 *), R (hydraulic radius) (r = 0.811 *) and P (unit stream power) (r = 0.811 *) in the moderate rainfall experiment. The results of this study will help guide further examination of the processes involved in the dynamic mechanisms of rill erosion on slopes under short-term heavy rainfall conditions.

Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis.

Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2-7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.

Discovery and Biosynthesis of Pepticinnamins G-M Featuring Three Enzymes-Catalyzed Nonproteinogenic Amino Acid Formation.

Since pepticinnamin E was discovered almost 30 years ago, no other pepticinnamin family of natural products has been reported to date. Here, we report the discovery of pepticinnamins G-I (1-3) from a marine Streptomyces sp. PKU-MA01144 and pepticinnamins J-M (4-7) from several mutants, and these new compounds contain different N-methyl-l-alanine and l-tyrosine residues compared to pepticinnamin E. Genome sequencing, heterologous expression, gene deletion, and reconstitution of enzymatic reaction in vitro identified the biosynthetic gene cluster of 1-7 and first experimentally established the biosynthesis of the nonproteinogenic 2-chloro-3-hydroxy-4-methoxy-l-phenylalanine residue by a biopterin-dependent hydroxylase Pep10, an O-methyltransferase Pep9, and a flavin-dependent halogenase Pep1. The biosynthetic research and heterologous expression system in this study set the stage for pathway engineering for more pepticinnamins generation in the future.

Discovery of a Phenylamine-Incorporated Angucyclinone from Marine Streptomyces sp. PKU-MA00218 and Generation of Derivatives with Phenylamine Analogues.

A new phenylamine-incorporated angucyclinone (1) featuring a unique 1-phenylbenzo[ cd]indol-3(1 H)-one moiety was discovered from marine Streptomyces sp. PKU-MA00218. A series of experimental investigations identified that 1 was produced from the nonenzymatic conversion of a C-ring-cleaved angucyclinone (2) with phenylamine. Utilizing the nonenzymatic conversion, 18 phenylamine-incorporated angucyclinone derivatives with halogen, methyl, methoxy, and carboxy substitutions were efficiently generated under mild conditions. These results highlighted the impressive roles of nonenzymatic reactions in expanding the structural diversity of angucyclinones.