Bacterial cellobiose metabolism: An inquiry-driven, comprehensive undergraduate laboratory teaching approach to promote investigative learning.

Technique-centered biochemistry or molecular biology undergraduate laboratory curricula do not offer significant opportunities for thoughtful, in-depth exploration of the science to foster investigative learning. To demonstrate inclusion of inquiry-driven laboratory experiments into the undergraduate biochemistry and molecular biology curricula, a comprehensive set of laboratory experiments, covering several principles of biochemistry and molecular biology, have been developed under a single theme. The laboratory curriculum described here comprehensively investigates bacterial cellobiose metabolism using multiple biochemical, molecular biological (RNA isolation, RT-PCR, PCR, and enzyme assay), and analytical techniques (High Performance Liquid Chromatography, NMR, spectrophotometry, and thin-layer chromatography) to explore the principles of metabolomics and genomics in a single undergraduate laboratory course setting using Caulobacter crescentus as the model organism.

Applicability of Instability Index for In vitro Protein Stability Prediction.

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method.

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus.

The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of β-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated.

Trapping and characterization of a reaction intermediate in carbapenem hydrolysis by B. cereus metallo-beta-lactamase.

Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem.

Folding strategy to prepare Co(II)-substituted metallo-beta-lactamase L1.

In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-beta-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.

Experimental evidence for a metallohydrolase mechanism in which the nucleophile is not delivered by a metal ion: EPR spectrokinetic and structural studies of aminopeptidase from Vibrio proteolyticus.

Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety.

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA.

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA.

Probing substrate binding to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis.

Background: The metallo-beta-lactamases are Zn(II)-containing enzymes that hydrolyze the beta-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-beta-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics.