Physiological temperature drives TRPM4 ligand recognition and gating.

Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.

Bidirectional Ventricular Tachycardia in a Women with Dilated Cardiomyopathy: A Case Report.

INTRODUCTION: Bidirectional ventricular tachycardia (BVT) is a rare arrhythmia characterized by QRS complexes with an axis in the frontal plane alternating polarity in the precordial leads and right bundle branch block (RBBB) morphology. To the best of our knowledge, there is no previous report in dilated cardiomyopathy or in the context of a probable peripartum cardiomyopathy. CASE PRESENTATION: A 26-year-old, 9-month female patient, with no significant past medical history (the patient denies medication intake, herbs like aconite, trouble during delivery, any heart issues or family history of sudden death or cardiomyopathies) who presents to the emergency room due to 11 days of dyspnea, exacerbated by daily activities, orthopnea, and paroxysmal nocturnal dyspnea. She presented with ventricular bigeminy and systolic dysfunction with left ventricular ejection fraction (LVEF) <20%. The patient later developed a stable ventricular tachycardia (VT) treated with amiodarone, which resulted in hemodynamic instability and BVT rhythm with VT paroxysms. Without the possibility of ablation, the use of high dose beta blockers and an implantable cardioverter defibrillator resulted in the same rhythm with a lower heart rate, better NYHA functional class, and less episodes of VTs. CONCLUSION: BVT is a rare type of tachycardia that can be present in dilated cardiomyopathy. In the absence of ablation capabilities, decreasing the heart rate with beta-blockers may reduce the rate of paroxysmal VTs.

Hypertensive ED patients: Missed opportunities for addressing hypertension and facilitating outpatient follow-up.

OBJECTIVES: Hypertension is a leading cause of morbidity and mortality. The emergency department (ED) frequently serves populations with unmet health needs and could have a greater and more systematic role in secondary prevention for hypertension. This study sought to determine, among hypertensive patients discharged from the ED, the frequency that patients 1) received hypertension-specific education, and 2) followed-up with a primary care provider. We secondarily assessed participant beliefs about hypertension. METHODS: This non-experimental, observational study enrolled a convenience sample of consenting patients with asymptomatic, markedly elevated blood pressure (systolic ≥160 mmHg or diastolic ≥100 mmHg) with medium to low triage acuity discharged from an urban, academic ED. Discharge instructions were assessed through chart review. Patients followed up per their normal routine without intervention. Participants were interviewed by phone two to four weeks after ED discharge to ascertain outpatient follow-up and describe beliefs about hypertension. RESULTS: From April through June 2014, 200 patients were approached, of whom 90 were enrolled. Of these, 77% of patients reported a previous diagnosis of hypertension, and 60% reported current treatment with antihypertensive medications. Five patients (5.5%) received written instructions at discharge addressing hypertension, although 59 (65.6%) reported that they were informed about their elevated blood pressure during the ED visit. Follow-up with a primary care provider within 2-4 weeks of discharge was completed in 57% of cases. None of the patients who received hypertension-specific discharge instructions completed follow-up. CONCLUSIONS: Over half of markedly hypertensive patients discharged from the ED followed up with primary care within four weeks. Nonetheless, missed opportunities for improved secondary prevention among ED patients with hypertension are common. There is an urgent need for evidence-based interventions to assist emergency departments in addressing this health threat.

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over ß-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the ß-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase.

In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-µL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

Whole-cell biocatalytic production of variously substituted ß-aryl- and ß-heteroaryl-ß-amino acids.

Biologically-active ß-peptides and pharmaceuticals that contain key ß-amino acids are emerging as target therapeutics; thus, synthetic strategies to make substituted, enantiopure ß-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼ 35) engineered to express a Pantoea agglomerans phenylalanine aminomutase (PaPAM) biocatalyst. In either 5 mL, 100mL, or 1L of M9 minimal medium containing α-phenylalanine (20mM), the cells produced ∼ 1.4 mg mL(-1) of ß-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted α-arylalanines to their (S)-ß-aryl-ß-amino acids in vivo and were not toxic to cells at mid- to late-stage growth. The ß-aryl-ß-amino acids made ranged from 0.043 mg (p-nitro-ß-phenylalanine, 4% converted yield) to 1.2mg (m-bromo-ß-phenylalanine, 96% converted yield) over 6h in 5 mL. The substituted ß-amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design.

A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-ß-Tyrosine with Unique High Enantioselectivity and Retention of Configuration.

A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-ß-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 µM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-ß- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(ß) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants.