Crystallization and preliminary X-ray analysis of the recombinant dihaem cytochrome c (NapB) from Haemophilus influenzae.

The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase (Nap). Recombinant NapB was overproduced in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop method. Thin quadrilateral plates were grown under various conditions but proved to be unsuitable for X-ray analysis. However, a single crystal was grown using 1.75 M ammonium sulfate in 0.1 M sodium acetate pH 5.5, from which a native data set could be collected to 1.8 A resolution using synchrotron radiation. Using the same conditions, further crystals were obtained by microseeding. The space group was determined to be P42(1)2, with unit-cell parameters a = 77.55, b = 77.55, c = 28.64 A and an unusually low solvent content of 16.5%, assuming there to be one molecule of NapB in the asymmetric unit. Analysis of the dissolved crystals indicated that partial proteolysis of the protein had occurred. Taking the molecular mass of the crystallized form ( approximately 8500 Da) into account, the solvent content was estimated to be 53%, with a V(M) value of 2.64 A(3) Da(-1).

Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis.

The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.

Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus.

An extracellular beta-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa; only the trimer was optimally active at 80 degrees C and at pH 4.5. At 90 degrees C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 degrees C for 48 h, but stability decreased above 70 degrees C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta-d-glucosides and cello-oligosaccharides, and was specific for substrates with a beta-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest K(m) towards p-nitrophenyl beta-d-glucoside (0.1137 mM) and the highest k(cat) towards cellobiose and beta,beta-trehalose (17052 min(-1)). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-delta-gluconolactone inhibited the beta-glucosidase competitively, with K(i) values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl beta-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that beta-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.