Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for activity. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481. KeywordsLantibiotic; leader peptide; lacticin 481; mutacin II; nukacin ISK-1The problem of multi-drug resistant bacteria has become increasingly apparent in recent years, with several strains posing the threat of becoming immune against all commercially available antibiotics (1,2). It is evident that in order to prevent potential epidemic outbreaks of infectious diseases, a renewed focus on antibiotic research is highly desired, including the understanding of biosynthetic pathways of natural product antibiotics (3). In this regard, the lantibiotics family of antimicrobial peptides has shown promising properties (4). Nisin, the most studied lantibiotic to date, is produced by Lactococcus lactis and has been used commercially as a preservative in the food industry for over 40 years due to its potent antibacterial properties and non-toxicity to humans (5). The high efficacy of nisin (nM MICs against many Gram-positive † This work was supported by the National Institutes of Health (GM58822 to WAV) and a Ruth L. Kirschstein National Research Service Award (GM070421 to LEC). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript bacteria) has been attributed ...

Lantibiotics are peptide antimicrobials containing the thioether-bridged amino acids lanthionine (Lan) and methyllanthionine (MeLan) and often the dehydrated residues dehydroalanine (Dha) and dehydrobutyrine (Dhb). While biologically advantageous, the incorporation of these residues into peptides is synthetically daunting, and their production in vivo is limited to peptides containing proteinogenic amino acids. The lacticin 481 synthetase LctM offers versatile control over the installation of dehydro amino acids and thioether rings into peptides. In vitro processing of semisynthetic substrates unrelated to the prelacticin 481 peptide demonstrated the broad substrate tolerance of LctM. Furthermore, a chemoenzymatic strategy was employed to generate novel thioether linkages by cyclization of peptidic substrates containing the nonproteinogenic cysteine analogs homocysteine and beta-homocysteine. These findings are promising with respect to the utility of LctM toward preparation of conformationally constrained peptide therapeutics.

IMP dehydrogenase (IMPDH) and GMP reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH/GMPR family have not been identified. Here, we show that the GMPR reaction utilizes the same intermediate E-XMP* as IMPDH, but this intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimic a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations, an "in" conformation poised for hydride transfer, and an "out" conformation where the cofactor is 6 Å from IMP. Mutagenesis, substrate/cofactor analog experiments demonstrate that the “out” conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery activating ammonia.

Lantibiotics are post-translationally modified antimicrobial peptides containing multiple cyclic thioethers that constrain their conformational freedom and that are required for their potent antimicrobial activities. 1 These crosslinks are installed in a two step process that involves dehydration of Ser and Thr residues to the corresponding dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, and the subsequent conjugate addition by cysteine thiols onto the dehydro amino acids (eg Figure 1A for lacticin 481). The ribosomally synthesized prepeptides contain an N-terminal leader peptide that is not modified during maturation and a C-terminal structural region that is transformed into the lantibiotic. The role of the leader peptide has been the subject of much speculation. Previous studies have shown that its removal is required for biological activity. 2,3 These findings suggest that it might play a protective role for producing organisms as the leader peptide is proteolytically removed in the final step of biosynthesis. Alternatively, the leader peptide may provide a recognition sequence for either the protease or the transporter that secretes the product. Finally, the leader peptide might function as a scaffold for the biosynthetic enzymes that generate the crosslinks because several recent studies have shown that lantibiotic dehydratases can process non-lantibiotic substrates attached at the C-terminus of a leader peptide. 4-6 The recent in vitro reconstitution of the activities of the biosynthetic enzymes for nisin, haloduracin, and lacticin 481 3 allows detailed investigation of the role of the leader peptide in the post-translational modifications. In this study we show that, surprisingly, the leader peptide is important but not required for dehydration by lacticin 481 synthetase (LctM).LctM tolerates a wide range of nonproteinogenic amino acids incorporated into the substrate peptide by expressed protein ligation. 6,7 While this approach allowed investigation of the substrate specificity of LctM, scale-up to produce the amounts required for quantitative SARstudies of lacticin 481 analogues proved difficult. We therefore investigated an alternative route to LctA analogues containing nonproteinogenic amino acids. A recent report showed that a substrate with three Ala residues inserted between the leader peptide and structural region of LctA was fully processed by LctM. 6 This observation prompted the evaluation of the nonpeptidic linkers 1 and 2 between the C-and N-terminal regions of LctA. In an initial test experiment, peptide 3 was obtained via the copper(I)-catalyzed [3+2] cycloaddition 8 of peptides 4 and 5. 9 Incubation of peptide 3 with LctM, ATP and Mg 2+ resulted in fourfold dehydration ( Figure 2A). Surprisingly, when peptides 4 and 5 were incubated with LctM to test enzymatic templation of the cycloaddition reaction, cycloaddition did not take place but partially dehydrated products were observed. A similar result was obtained when a heterologously expressed, His 6 -tagged LctA analog was cleav...