CT attenuation values are highly correlated with chemical-shift ratios. Both values were indeterminate for a similar subset of benign lesions.

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5'-phosphate (PLP)-dependent enzyme (Fub7) and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic alpha,beta-unsaturated aldehyde. By harnessing the stereoselective C-C bond forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl, 5,5-dialkyl, and 5,5,6-trialkyl-L-pipecolic acids of high diastereomeric ratio. File list (3) download file view on ChemRxiv FubManuscript_SI_20200830.docx (13.26 MiB) download file view on ChemRxiv YH_Fub_MS_20200830_article_ed2.pdf (1.00 MiB) download file view on ChemRxiv YH_Fub_MS_20200830_article_ed2.docx (1.29 MiB) Supporting InformationBiosynthesis of mycotoxin fusaric acid and application of a PLP-dependent enzyme for chemoenzymatic synthesis of substituted L-pipecolic acids.

A nonribosomal peptide synthetase (NRPS)-nonreducing polyketide synthase (NRPKS) hybrid enzyme (AnATPKS) from Aspergillus niger was shown to produce amino acid derived α-pyrone natural products (pyrophen and campyrone B). Biochemical characterization of the NRPS module in vitro reveals that the adenylation domain is promiscuous toward a variety of substituted phenylalanine analogues. Using precursor feeding and heterologous expression of AnATPKS and an associated O-methyltransferase (AnOMT), we were able to access a library of substituted pyrophen analogues. Our study paves the way for future combinatorial biosynthesis of diverse α-pyrone natural products using NRPS–NRPKS hybrids.

Nonribosomal peptide synthetases (NRPSs) and NRPS-like enzymes have diverse functions in primary and secondary metabolisms. By using a structure-guided approach, we uncovered the function of a NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on the homology model suggests cation-π interactions as the major substrate specificity determinant, which was verified using substrate analogs and inhibitors. Bioinformatic analysis indicates this NRPS-like glycine betaine reductase is highly conserved and widespread in kingdom fungi. Genetic knockout experiments confirmed its role in choline biosynthesis and maintaining glycine betaine homeostasis in fungi. Our findings demonstrate that the oxidative choline-glycine betaine degradation pathway can operate in a fully reversible fashion and provide insight in understanding fungal choline metabolism. The use of an NRPS-like enzyme for reductive choline formation is energetically efficient compared with known pathways. Our discovery also underscores the capabilities of the structure-guided approach in assigning functions of uncharacterized multidomain proteins, which can potentially aid functional discovery of new enzymes by genome mining.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane a helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first a helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is conduded that Trp-33 may be involved directly or indirectly in sugar recognition.In recent years, it has been possible to study the correlation between structure at the amino acid level and the function of membrane transport proteins. At present, there is a great deal of data available on specific mutants of the lactose carrier of Escherichia coli. More than 150 mutants have been isolated and studied for various aspects of transport. Two general techniques have been used to generate these mutants. The first of these has been the isolation of spontaneous mutants by using a variety of special selection techniques (1-3, 16). The second method is that of site-directed mutagenesis (11,17,22) or cassette mutagenesis (9). Mutants derived from these procedures have been extremely useful in determining amino acid residues which are essential for normal transport function and may be involved in either sugar recognition or coupling of cation movement to sugar transport (for reviews, see references 1 and 22).A useful variation on the point mutagenesis theme involves the use of natural as well as synthetic amber suppressor strains (13,(18)(19)(20)