PubMed · 7633519
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K Kuwajima. 1995. Circular dichroism.. https://doi.org/10.1385/0-89603-301-5%3A115
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Microbes produce bioactive secondary metabolites as toxins, pigments, or virulence factors. These specialized compounds are produced by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrid NRPS/PKS pathways. The genes encoding NRPS and PKS reside in biosynthetic gene clusters (BGCs), some of which have no identified metabolite associated with them. Characterization of these orphan BGCs could provide insights into potential bioactive compounds that have yet to be discovered. Here, we characterize PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1. We cloned, expressed, and purified PA1216, and developed an optimized differential scanning fluorimetry assay to measure its thermal stability, demonstrating concentration-dependent stabilization in the presence of established methyltransferase cofactors and inhibitors. We then adapted this assay for high-throughput screening of potential PA1216 substrates, identifying destabilizing compounds, including glycyl-glycine dipeptides, amino esters with aromatic or basic side chains, and N-Boc-protected amino acids. In contrast, sodium salts of organic acids stabilized PA1216. Lastly, we employed AlphaFold to construct a predictive model, revealing that PA1216 contains a Rossmann-like fold and a glycine-rich loop, typical of class I methyltransferases, and we corroborated these secondary structural elements using circular dichroism spectroscopy. Overall, these studies illuminate PA1216 function and establish a platform for characterizing cryptic gene clusters within secondary metabolic pathways.
Bacillus stearothermophilus phosphoglycerate kinase (bsPGK) is a monomeric enzyme of 394 residues comprising two globular domains (N and C), covalently linked by an interdomain alpha-helix (residues 170-185). The molecule folds to the native state in three stages. In the first, each domain rapidly and independently collapses to form an intermediate in which the N-domain is stabilized by 5.1 kcal mol-1 and the C-domain by 3.3 kcal mol-1 over their respective unfolded conformations. The N-domain then converts to a folded state at a rate of 1.2 s-1 (delta GI-F = 3.8 kcal mol-1), followed by the C-domain at 0.032 s-1 (delta GI-F = 12.1 kcal mol-1). It is this last step that limits the rate of acquisition of enzyme activity. In the dynamics of unfolding in water, the N-domain converts to the intermediate state at a rate of 8 x 10(-4) s-1, some 10(7) times faster than the C-domain. Consequently, the most populated intermediate in the folding reaction has a native-like N-domain, while that in the unfolding direction has a native-like C-domain. In a conventional sense, therefore, the folding/unfolding kinetics of bsPGK can be described as random order. Consistent with these observations, cutting the molecule in the interdomain helix produces two, independently stable units comprising residues 1-175 and 180-394. A detailed comparison of their folding behavior with that of the whole molecule reveals that true interdomain contacts are relatively weak, contributing approximately 1.4 kcal mol-1 to the stability of the active enzyme. The only interactions which contribute to the stability of rapidly formed intermediates or to transition states along the productive folding pathways are those within domain cores. Contacts formed either between domains or with the interdomain helix are made only in the folded ground state, but do not constitute a separate step in the folding mechanism. Intriguingly, the most pronounced effect of interdomain contacts on the kinetics of folding is inhibitory; the presence of the C-domain appearing to reduce the effective rate of acquisition of native structure within the N-domain.
DNA oligonucleotide sequences containing two parallel complementary strands attached through 3'-3' and 5'-5' linkages were synthesized. These oligonucleotides from Hoogsteen base-paired parallel-stranded (PS) hairpin duplexes under appropriate conditions [Kandimalla, E. R., Agrawal, S., Venkataraman, G., & Sasisekharan, V. (1995a) J. Am. Chem. Soc. 117, 6416-6417]. UV melting experiments show that these Hoogsteen hairpin duplexes have a lower thermal stability than that of the Watson-Crick (WC) hairpin duplex (antiparallel) of the same sequence. The circular dichroism (CD) spectrum of the Hoogsteen duplex is different from the canonical B-DNA WC duplex spectrum. The formation of the Hoogsteen duplex is pH-dependent since protonation of cytosine requires lower pH conditions. Studies with oligonucleotides of different loop sizes revel that three- and two-base loops are optimum for the formation of stable Hoogsteen duplexes with 3'-3' and 5'-5' linkages, respectively. The guanine residues in the loop stabilize the duplex as a result of G-G interactions as confirmed by molecular modeling studies. The new PS Hoogsteen duplexes form stable triplexes with complementary (antiparallel to the purine domain) single-stranded RNA and DNA pyrimidine sequences in Py.Pu:Py (pyrimidine third strand-purine WC strand:pyrimidine WC strand) motif. The thermal stability of the resulting triplexes is much higher than that of the conventional triplex (binding of a Hoogsteen pyrimidine third strand to a WC duplex) of the same sequence. The CD spectra of the new triplexes are similar to those of conventional triplexes, suggesting that no conformational change occurs as a result of 3'-3' or 5'-5' linkage. A molecular modeling study was carried out to examine the stereochemical feasibility of the Hoogsteen duplexes and formation of triplexes with single-stranded pyrimidine complementary strands.