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Aminoacyl-tRNA synthetases should ensure high accuracy in tRNA aminoacylation. However, the absence of significant structural differences between amino acids always poses a direct challenge for some aminoacyl-tRNA synthetases, such as leucyl-tRNA synthetase (LeuRS), which require editing function to remove mis-activated amino acids. In the cytoplasm of the human pathogen Candida albicans, the CUG codon is translated as both Ser and Leu by a uniquely evolved CatRNASer(CAG). Its cytoplasmic LeuRS (CaLeuRS) is a crucial component for CUG codon ambiguity and harbors only one CUG codon at position 919. Comparison of the activity of CaLeuRS-Ser919 and CaLeuRS-Leu919 revealed yeast LeuRSs have a relaxed tRNA recognition capacity. We also studied the mis-activation and editing of non-cognate amino acids by CaLeuRS. Interestingly, we found that CaLeuRS is naturally deficient in tRNA-dependent pre-transfer editing for non-cognate norvaline while displaying a weak tRNA-dependent pre-transfer editing capacity for non-cognate α-amino butyric acid. We also demonstrated that post-transfer editing of CaLeuRS is not tRNALeu species-specific. In addition, other eukaryotic but not archaeal or bacterial LeuRSs were found to recognize CatRNASer(CAG). Overall, we systematically studied the aminoacylation and editing properties of CaLeuRS and established a characteristic LeuRS model with naturally deficient tRNA-dependent pre-transfer editing, which increases LeuRS types with unique editing patterns.

The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield.

Occurrence of toxigenic molds and mycotoxins on dried fruits is a worldwide problem, but limited information is available in China. A total of 220 dried fruits (raisins, dried apricots, dates and wolfberries) purchased from China were analyzed for 17 mycotoxins (i.e., Alternaria toxins, ochratoxin A (OTA), patulin (PAT) and trichothecenes) by UPLC-MS/MS, combined with a single-step cleanup. The result showed that at least one mycotoxin was detected in 142 samples (64.6%). The lowest incidence of contaminated samples was observed in dried apricots (48.2%), and the highest incidence in dried wolfberries (83.3%). The Alternaria toxins seemed to be the major problem in dried fruits, rather than OTA or PAT. Tenuazonic acid (TeA) was the predominant mycotoxin, in both frequency and concentration, ranging from 6.9 to 5665.3 μg kg−1, followed by tentoxin (TEN; 20.5%), and mycophenolic acid (MPA; 19.5%). Moreover, raisins are more likely to be contaminated with OTA than the other dried fruits. Penicillic acid (PA) was detected only in dried dates, and PAT was detected only in one apricot sample. In addition, our results also showed that the simultaneous presence of 2–4 mycotoxins was observed in 31.4% of dried fruits. TeA and TEN were the most frequent combination, detected in 29 (13.2%) samples, followed by TeA and MPA with a prevalence of 11.4%. Therefore, the results of this survey suggest the need for wider monitoring on the contamination of these mycotoxins, especially Alternaria toxins in agro-products, and indicate the importance of setting a maximum limit for Alternaria toxins in China.