Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK.

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.

In-depth Sequence-Function Characterization Reveals Multiple Pathways to Enhance Enzymatic Activity.

Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence-function relationships but it has not been well-explored as a guide to enzyme engineering and identifying pathways by which their catalytic cycle may be improved. We report such a demonstration in this work using a Phenylalanine ammonia-lyase (PAL), which deaminates L-phenylalanine to trans-cinnamic acid and has widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis (AvPAL*) has garnered significant attention as the active ingredient in Pegvaliase®, the only FDA-approved drug treating classical Phenylketonuria (PKU). Although an extensive body of literature exists on the structure, substrate-specificity, and catalytic cycle, protein-wide sequence determinants of function remain unknown, as do intermediate reaction steps that limit turnover frequency, all of which has hindered rational engineering of these enzymes. Here, we created a detailed sequence-function landscape of AvPAL* by performing DMS and revealed 112 mutations at 79 functionally relevant sites that affect a positive change in enzyme fitness. Using fitness values and structure-function analysis, we picked a subset of positions for comprehensive single- and multi-site saturation mutagenesis and identified combinations of mutations that led to improved reaction kinetics in cell-free and cellular contexts. We then performed QM/MM and MD to understand the mechanistic role of the most beneficial mutations and observed that different mutants confer improvements via different mechanisms, including stabilizing transition and intermediate states, improving substrate diffusion into the active site, and decreasing product inhibition. This work demonstrates how DMS can be combined with computational analysis to effectively identify significant mutations that enhance enzyme activity along with the underlying mechanisms by which these mutations confer their benefit.

Small and potentially resectable focal pancreatic lesions noted on CT/MRI scans in nonjaundiced patients: likelihood of neoplasia and utility of EUS.

BACKGROUND: Management of small and potentially resectable "mass" lesions encountered on CT/MRI scans in patients without obstructive jaundice (ObJ) is rather empirical since there is scant data on likelihood of neoplasm to formulate treatment strategies. We evaluated (1) the prevalence of neoplasm and (2) performance characteristics of EUS-FNA for diagnosing neoplasm in above-mentioned subset of patients. PATIENTS: This is a retrospective analysis of 232 patients (without ObJ) with a focal pancreatic lesion, ≤ 25 mm and potentially resectable on CT/MRI who underwent EUS-FNA from 2002 to 2009. RESULTS: Seventy-five patients (32.3%, 95% CI 26.6, 38.6) were finally diagnosed to have a neoplasm. Four of 92 (4.3%) lesions ≤ 15 mm, 13 of 57(22.8%) lesions 16-20 mm, and 35 of 83 (42.1%) lesions 21-25 mm had an adenocarcinoma. Larger lesion size, older patient age, and h/o recent weight loss significantly increased the likelihood of adenocarcinoma. EUS-FNA had 98.2% overall accuracy and 98.1% NPV with no significant differences based on lesion size. CONCLUSIONS: In nonjaundiced patient with a potentially resectable pancreatic lesion ≤25 mm in size noted on CT/MRI scanning, EUS-FNA can provide useful adjunctive information to optimize the use of surgery and can potentially obviate the need for "wait and watch approach" with repeat imaging in their clinical management.

Diagnostic value of EUS-FNA in patients suspected of having pancreatic cancer with a focal lesion on CT scan/MRI but without obstructive jaundice.

OBJECTIVE: Patients frequently present with suspected pancreatic neoplasm based on a focal pancreatic lesion on computed tomographic (CT) scan/magnetic resonance image (MRI) but without obstructive jaundice. We evaluated the performance characteristics of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in this patient subset. METHODS: This is a retrospective analysis of a prospective database and included patients who underwent EUS-FNA at a university hospital for a focal pancreatic lesion noted on CT/MRI. Patients were excluded if (1) they had obstructive jaundice or (2) the lesion appear (seem)ed cystic on CT/MRI. The main outcome measurements were (1) prevalence of pancreatic cancer and (2) performance characteristics of EUS-FNA for identifying malignancy. RESULTS: In the 213 study patients, a focal pancreatic lesion was identified in 173 patients by EUS. The final diagnosis included adenocarcinoma (n=89), neuroendocrine tumor (n=14), mucinous cystadenocarcinoma (n=1), solid pseudopapillary tumor (n=2), metastases (n=4), benign cyst (n=19), pseudocyst (n=9), abscess (n=4), chronic pancreatitis (n=32), and normal pancreas (n=39). Endoscopic ultrasound-guided FNA had an accuracy of 97.6% for diagnosing malignant neoplasm, with 96.6% sensitivity, 99.0% specificity, 96.2% negative predictive value, and 99.1% positive predictive value. CONCLUSIONS: Endoscopic ultrasound-guided FNA is highly accurate for diagnosing malignancy in patients with a focal pancreatic lesion on CT scan/MRI but without obstructive jaundice. Endoscopic ultrasound-guided FNA can potentially be used as a definitive diagnostic test in the management of these patients.

EUS/EUS-FNA for suspected pancreatic cancer: influence of chronic pancreatitis and clinical presentation with or without obstructive jaundice on performance characteristics.

BACKGROUND: The clinical utility of EUS-FNA is debated in patients with obstructive jaundice (ObJ) because of a very high pretest probability of pancreatobiliary malignancy (PBM) and biliary stent-induced inflammation that can potentially confound EUS-FNA diagnosis. EUS-FNA also has lower accuracy in patients with underlying chronic pancreatitis (CP). OBJECTIVE: Our purpose was to determine the clinical value of EUS-FNA for PBM diagnosis based on clinical presentation and presence of CP. DESIGN: Retrospective analysis of prospective database. SETTING: University hospital. PATIENTS: Patients who underwent EUS-FNA from 2002 to 2006 for suspected PBM based on (1) ObJ with biliary stricture or a mass lesion or (2) abnormal pancreatic imaging by CT/MRI: a focal pancreatic "mass" lesion; dilated pancreatic duct +/- common bile duct; or an enlarged head of pancreas. INTERVENTIONS: EUS was performed with a radial echoendoscope followed by a linear echoendoscope if a focal pancreatic lesion was identified. Fine-needle aspirates were assessed immediately by an attending cytopathologist. MAIN OUTCOME MEASUREMENTS: (1) Prevalence of cancer and (2) performance characteristics of EUS-FNA. RESULTS: PBM was diagnosed in 73.9% of patients with ObJ and biliary stricture or pancreatic mass, in 49.6% of patients with pancreatic mass, and in 7.0% of patients with an enlarged head of pancreas or dilated pancreatic duct +/- common bile duct. The prevalence of PBM was lower in all 3 presentations with associated CP. Both CP and presentation with ObJ lowered performance characteristics of EUS-FNA, but CP did so only in the subset of patients with ObJ. All except 1 false-negative diagnoses were due to cytologic misinterpretation. LIMITATION: Retrospective design. CONCLUSION: Among patients with suspected PBM, the accuracy of EUS-FNA is significantly lower only in a subset of patients with ObJ with underlying CP, largely as a result of difficulty in cytologic interpretation.

EUS and/or EUS-guided FNA in patients with CT and/or magnetic resonance imaging findings of enlarged pancreatic head or dilated pancreatic duct with or without a dilated common bile duct.

BACKGROUND: Incidental findings of an enlarged head of pancreas (HOP) or dilated pancreatic duct (PD) with or without a dilated common bile duct (CBD) on CT or magnetic resonance imaging (MRI), in patients without obstructive jaundice, raise suspicion for a pancreatic neoplasm, but their clinical significance has not been established. OBJECTIVE: To determine the prevalence of pancreatic neoplasm in this patient group. DESIGN: Retrospective analysis of a prospective database. SETTING: Tertiary-care university hospital. PATIENTS: Patients without obstructive jaundice at initial presentation, who underwent EUS and/or EUS-guided FNA (EUS-FNA) for an abnormal CT and/or MRI with an enlarged HOP (n = 67) or a PD with or without a dilated CBD (n = 43). The final diagnosis was based on definitive cytology, surgical pathology, and clinical follow-up. INTERVENTIONS: An EUS examination was performed by using a radial echoendoscopy followed by a linear echoendoscopy, if a focal pancreatic lesion was identified. Fine-needle aspirates were stained with Diff-Quik and Papanicolaou's methods, and were immediately assessed by an attending cytopathologist. MAIN OUTCOME MEASUREMENTS: (1) The prevalence of pancreatic neoplasms and (2) performance characteristics of EUS-FNA for identifying malignant neoplasm, in this patient group. RESULTS: In 110 study patients, the final diagnosis included adenocarcinoma (n = 7), pancreatic intraepithelial neoplasia (n = 1), neuroendocrine tumor (n = 1), tumor metastasis (n = 1), and benign cyst (n = 3). Thirty-two patients had EUS evidence of chronic pancreatitis, and, in the remaining 65 patients, the pancreas was normal. The accuracy of EUS and EUS-FNA for diagnosing pancreatic neoplasm in these patients was 99.1%, with 88.8% sensitivity, 100% specificity, 99% negative predicative value, and 100% positive predictive value. LIMITATION: A retrospective design and surgical confirmation in only a small number of study patients. CONCLUSION: A pancreatic neoplasm is seen in a clinically significant number of patients with "enlarged HOP" or "dilated PD with or without a dilated CBD" but without obstructive jaundice. EUS-FNA seems highly accurate for diagnosing pancreatic neoplasm in these patients.

Intraductal US in evaluation of biliary strictures without a mass lesion on CT scan or magnetic resonance imaging: significance of focal wall thickening and extrinsic compression at the stricture site.

BACKGROUND AND OBJECTIVE: The clinical utility of intraductal US (IDUS) for evaluating biliary strictures has been limited because of a lack of easily recognized morphologic criteria to distinguish benign and malignant strictures. We studied the clinical value of 2 easily assessed IDUS findings: wall thickness and extrinsic compression at the stricture site. DESIGN AND SETTING: A retrospective, single-center study. PATIENTS AND METHODS: Forty-five patients without an identifiable mass on CT/magnetic resonance imaging, who underwent ERCP/IDUS for evaluation of biliary strictures were studied. IDUS pictures were reviewed specifically to measure wall thickness and to look for extrinsic compression at the stricture site. MAIN OUTCOME MEASUREMENTS AND RESULTS: The mean age of the patients was 64.2+/-13.3 years. Thirty patients had jaundice at presentation, and in 15 patients a stricture was suspected on imaging. The mean length of biliary strictures was 15.1+/-7.8 mm. Strictures were distal (distal common bile duct) in 25 patients and proximal (mid/proximal common bile duct or common hepatic duct) in 20 patients. Fourteen strictures were finally diagnosed to be malignant. Strictures in 20 patients were caused by extrinsic compression, and tissue diagnosis was readily obtained by EUS-FNA in all these patients. Of 25 strictures without extrinsic compression, 6 were malignant (wall thickness 9-16 mm) and 19 were benign (wall thickness

Periportal lymphadenopathy in patients without identifiable pancreatobiliary or hepatic malignancy.

BACKGROUND & AIMS: Enlarged periportal lymph nodes often are noticed during imaging of the upper abdomen. Malignant infiltration and enlargement of periportal nodes occur in patients with cancers of the liver, gallbladder, biliary tree, and pancreas and lymphoma. However, there are no published data on the significance and differential diagnosis of enlarged periportal lymph nodes in patients without the above mentioned cancers. METHODS: We searched our database for patients who (1) underwent endoscopic ultrasound for evaluation of enlarged periportal nodes or (2) were found to have enlarged periportal lymph nodes (> or =10 mm) during endoscopic ultrasound (EUS) examination. Patients with identifiable pancreatic, biliary, gallbladder, or liver cancers were excluded. EUS-guided fine-needle aspiration of one or more nodes was performed. RESULTS: Sixty-four patients with periportal lymph nodes 10-40 mm in size met the inclusion criteria. In 24 patients, enlarged periportal nodes were noted in the computerized tomography or magnetic resonance imaging scans. Fifty-one patients had multiple enlarged periportal nodes. Concomitantly, enlargement was seen in peripancreatic nodes (n = 14), celiac nodes (n = 14), and mediastinal nodes (n = 11). Twelve of the 64 patients (18.8%; 95% confidence interval, 9.2%-28.4%) had a malignant cause of enlarged periportal lymph nodes: 5 with metastatic carcinoma and 7 with non-Hodgkin's lymphoma. Significant cytologic findings in benign nodes included granulomas (n = 4) and lipogranulomatosis (n = 8). CONCLUSIONS: A significant number of patients with enlarged periportal lymph nodes without identifiable pancreatobiliary and liver cancer harbor malignancy and other identifiable pathologic processes. We recommend that these nodes be sampled with fine-needle aspiration at the time of EUS examination.