Author index of volume 26, 2008

The stereospecific esterification of ibuprofen by Candida rugosa lipase (CRL) with 1-butanol was optimised. The yield of the butyl ester was nearly quantitative with an enantiomeric excess of 95% and E 100. Enzyme desiccation over P₂O₅ had a pronounced effect on the esterification yield and its role in stereospecificity is discussed. Molecular modelling and energy-minimisation studies were also performed to understand the stereospecific binding of substrates to the active site of CRL. The docking of the substrate S(+) ibuprofen to the active site of CRL was performed based on the three-dimensional structure of CRL (PDB entry 1CRL). The results show that only the active S(+) enantiomer of ibuprofen is able to form direct contacts with the reactive hydroxyl group (Ser209) and imidazole base (His449) at the active site, whereas with the R enantiomer the functional group COOH points away from the (His449) base of CRL. This is in accordance with experimental results which show that the stereospecifity of CRL is towards S(-) ibuprofen.     

Stereoisomers of cysteine and its analogs Potential effects on chemo- and radioprotection strategies

Summary The thiol-containing amino acid, cysteine, and its analogs are useful for a variety of protective applications, including protecting normal tissues against the unwanted side effects of cancer chemotherapeutic agents and radiation treatment. The protection can result from both direct action of the amino acid and/or after its conversion to glutathione (GSH), sulfate, or other sulfur-based protective substances. Unfortunately, high GSH levels have been implicated in the problematic development of tumor cells' resistance to therapy. Due to numerous differences in the metabolic processing of the cysteine stereoisomers, chemo- and radioprotective strategies might be developed using the D-form of the amino acid, which can participate in protection directly, but which cannot be used to support GSH biosynthesis. In this way, protection of normal tissue may be achieved, while the potential development of resistance in tumor cells is minimized. Greatly enhanced therapeutic efficacy of cancer treatment regimens may be the result.     

Purification and characterization of NADP-dependent 7β-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52

An NADP-dependent 7β-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7β-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (K_m) value of 0.05 mM for 3α,7β-dihydroxy-5β-cholanic acid whereas higher values were obtained with 3α,7β-dihydroxy-5β-cholanoyl glycine (0.20 mM), and 3α,7β-dihydroxy-5β-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and has a K_m value of 0.30 mM. A molecular weight of 64 000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.     

Enzymatic syntheses of ketoses: study and modification of the substrate specificity of the transketolase from Saccharomyces cerevisiae

Transketolase (TK) is a useful catalyst for ketose syntheses. The first part of this paper reports a convenient and easy method to synthesise 4-deoxy- -fructose-6-phosphate, potential inhibitor of sugar metabolism. TK used in synthetic purposes is the enzyme from Saccharomyces cerevisae, which is commercially available, or the enzyme from spinach leaves which we obtained as a crude extract. But these sources are expensive or give small quantities of the enzyme. In order to obtain larger amounts of enzyme, we use TK overexpressed in S. cerevisiae. The three-dimensional structure being known, the study and modification of the substrate specificity of this enzyme can be investigated by site-directed mutagenesis. In the second part of this paper, our study shows that Asp 477 is involved in determining the stereospecificity towards C2 hydroxyl group of the acceptor substrate.     

Incorporation of 18O2 and absence of stereospecificity in primary product formation during fungal metabolism of a lignin model compound

The metabolism of a lignin substructure model compound, 1,2-bis(3-methoxy-4-ethoxyphenyl)propane-1,3-diol (Ia) in ligninolytic cultures of Phanerochaete chrysosporium was studied to help elucidate the biochemical mechanism of lignin degradation. The primary reaction was cleavage of the model compound between C₁ and C₂ of the propane moiety to produce 1-(3-methoxy-4-ethoxyphenyl)ethane-1,2-diol and a C₆-C₁ product (probably 3-methoxy-4-ethoxybenzaldehyde). Other identified products arose secondarily; all were further metabolized. Even though the model compound was a mixture of four stereoisomers, no stereoselectivity was observed in its metabolism. In cultures under ¹⁸O₂, the initial cleavage produced the diol product with ≈70% enrichment by ¹⁸O in the benzyl alcohol group. The diol was a mixture of the two possible enantiomers, and the O₂-derived hydroxyl was incorporated at the asymmetric (benzyl) carbon. (Limited optical activity in the diol was traced to selective further metabolism of the D form.) These results show that the primary cleavage reaction lacked stereospecificity and was primarily oxygenative, implicating a nonspecific oxygenase or a nonenzymatic reaction involving activated oxygen. Preliminary experiments demonstrated no cell homogenate activity against Ia.