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A proper regulation of membrane fluidity is critical for cellular activities such as communication between cells, mitosis, and endocytosis. Unsaturated lipids, a main component of biological membranes, are particularly susceptible to oxidative attack of reactive oxygen species. The oxidation of lipids can produce structural derangement of membranes and eventually alter the membrane fluidity. We have applied fluorescence correlation spectroscopy (FCS) and Raman spectroscopy to investigate the fluidity and structure of model membranes subject to oxidative attack. Hydrogen peroxide has little effect on the lateral fluidity of membranes, whereas hydroxyl radical causes a significantly increased fluidity. The latter is rationalized with the cleavage of the acyl chains of lipids caused by hydroxyl radical; this interpretation is founded on the diminished intensities of lines in Raman spectra associated with -CH(2) and C═C moieties in lipids and supported by mass-spectral measurements. The same approach provides a mechanistic account of the inhibitory capability of vitamins C and E against the increased membrane fluidity resulting from an oxidative attack. Membranes with much cholesterol exhibit a novel resistance against altered membrane fluidity induced with oxidative attack; this finding has biological implications. Our approach combining FCS and Raman measurements reveals the interplay between the structure and fluidity of membranes and provides insight into the pathophysiology of cellular oxidative injury.

Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-β-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are β-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.

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Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.

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