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Interconversion of the androstenedione hydroxylase specificities of cytochromes p450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1996年第1期335卷 152-160页

作者： He, YQ Szklarz, GD Halpert, JR UNIV ARIZONA COLL PHARMDEPT PHARMACOL & TOXICOLTUCSONAZ 85721

Based on recent studies of single reciprocal mutants of cytochrome P450 2B4 and the highly related P450 2B5 at positions 114, 294, 363, and 367 [G. D. Szklarz, Y. Q. He, K. M. Kedzie, J. R. Halpert, and V. L. Burnett (1996) Arch. Biochem. Biophys. 327, 308-318], a number of multiple mutants were constructed, expressed in Escherichia coli, and assayed with androstenedione, progesterone, and benzyloxyresorufin. Simultaneous substitutions of Ile-114, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 with Phe, Thr, Val, and Ala, respectively from 2B5, resulted in a marked increase in androstenedione 15 alpha- and 16 alpha-hydroxylation compared with the wild-type enzyme and yielded a metabolite profile indistinguishable from that of cytochrome P450 2B5. Likewise, the reciprocal P450 2B5 quadruple mutant exhibited the specificity for 16 beta-hydroxylation characteristic of the 2B4 wild type. The two reciprocal quadruple mutants of P450 2B4 and 2B5 also displayed benzyloxyresorufin dealkylase activities similar to those of the wild-type P450 2B5 and 2B4, respectively. However, the progesterone metabolite profile of P450 2B5 was not identical to that of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue. Therefore, the 17 beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.

关键词： cytochrome P450 mutagenesis steroid hydroxylation structure-function relationships heterologous expression molecular modeling

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Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1996年第2期327卷 308-318页

作者： Szklarz, GD He, YQ Kedzie, KM Halpert, JR Burnett, VL UNIV ARIZONA COLL PHARM DEPT PHARMACOL & TOXICOL TUCSON AZ 85721 USA

The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation, Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile-114 --> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity. (C) 1996 Academic Press, Inc.

关键词： cytochrome P450 mutagenesis steroid hydroxylation structure-function relationships heterologous expression molecular modeling

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Peripheral and central predictors of whisker afferent morphology in the rat brainstem

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JOURNAL OF COMPARATIVE NEUROLOGY 1996年第3期375卷 481-501页

作者： Shortland, PJ Demaro, JA Shang, F Waite, PME Jacquin, MF WASHINGTON UNIV SCH MED DEPT NEUROL ST LOUIS MO 63110 USA UNIV NEW S WALES SCH ANAT SYDNEY NSW 2052 AUSTRALIA

Prior studies suggest that whisker afferents have but one central projection pattern, despite their association with differing peripheral receptors that predict central morphology in other systems. Target factors in barrelettes are thought to dictate afferent projection patterns;yet, barrelettes differ in their size, shape and development. We tested the hypothesis that whisker afferents have differing morphologies that are predicted by peripheral and central factors. Branching patterns and collaterals of 78 Neurobiotin-stained afferents were compared in rats. Fibers from one whisker had precisely somatotopic projections but highly varied morphologies. For the entire sample, analysis of variance revealed significant intrafiber variance in collateral number and arbor shape that was attributed to the target subnucleus. Significant interfiber variance did not reflect response adaptation rate, direction sensitivity, whisker row origin or parent fiber bifurcation in the trigeminal root. Instead, we found the following. 1) Mandibular fibers had more elongated arbors than maxillary axons. In subnuclei interpolaris and principalis, mandibular fibers had larger arbors with more boutons/collateral than maxillary axons;in oralis and interpolaris, mandibular fibers had fewer collaterals than those of the maxillary division. 2) Upper lip whisker axons had more boutons than those from the B-D row in all subnuclei. 3) Rostral whisker are afferents had larger arbors and more boutons than those from middle or caudal arcs due to significant are effects in interpolaris and oralis. Thus, whisker afferents are not structurally uniform, and some morphological features are predictable. Intrafiber variance is attributed to the central target;interfiber variance reflects maxillary versus mandibular origin, upper lip origin and whisker rostrocaudal are. (C) 1996 Wiley-Liss, Inc.

关键词： trigeminal barrel somatosensory structure-function relationships Neurobiotin

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Mechanisms of active transport in the F0F1 ATP synthase

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JOURNAL OF MEMBRANE BIOLOGY 1996年第2期151卷 101-111页

作者： Nakamoto, RK Department of Molecular Physiology and Biological Physics University of Virginia Jordan Hall Box 449 Charlottesville VA 22908 US

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Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II

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MOLECULAR & GENERAL GENETICS 1996年第1期250卷 1-6页

作者： KawagishiKobayashi, M Yamamoto, M Ishihama, A NATL INST GENET DEPT MOLEC GENETMISHIMASHIZUOKA 411JAPAN UNIV TOKYO SCH SCIDEPT BIOCHEM & BIOPHYSTOKYO 113JAPAN

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 of Schizosaccharomyces pombe was expressed in Escherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with P-32-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutant rpb2 alleles, rpb2(E357)A and rpb2(H386L), each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutant rpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutant rpb2 alleles were expressed in an rpb2-disrupted S. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.

关键词： RNA cleavage transcription RNA synthesis ribonuclease structure-function relationships

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Biological adhesion at interfaces

ANNUAL REVIEW OF MATERIALS SCIENCE

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ANNUAL REVIEW OF MATERIALS SCIENCE 1996年第1期26卷 651-691页

作者： Hammer, DA Tirrell, M UNIV MINNESOTA DEPT CHEM ENGN & MAT SCI MINNEAPOLIS MN 55455 USA

In biological adhesion at interfaces, the specific recognition between reactive biomacromolecules or receptors occurs against a backdrop of polymeric and long-range nonspecific forces. We identify the important functional parameters that characterize biomolecule function in adhesion, with an emphasis on receptors that mediate cell rolling. Receptors are the ultimate ''smart'' materials that can adjust their function in response to environmental changes or biochemical modification. Bioadhesion is often nonideal owing to dissipative processes or interfacial reorganization. Promising new developments to elucidate the functional properties of bioadhesion molecules are probes to measure forces on single adhesive bonds, genetic engineering, molecular reconstitution, computer simulation, and structural analysis and manipulation.

关键词： receptors ligands specific recognition biological membranes interfacial forces friction structure-function relationships

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Copper binding studies of lipases from different sources using image binding: Mechanism of free copper and chelated copper

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JOURNAL OF MOLECULAR RECOGNITION 1996年第5-6期9卷 715-721页

作者： Colin, C Vijayalakshmi, MA UNIV TECHNOL COMPIEGNE LAB INTERACT MOL & TECHNOL SEPARATF-60206 COMPIEGNEFRANCE

Immobilized metal-ion affinity gel electrophoresis (IMAGE) has been demonstrated to be an efficient tool to study the binding of proteins to chelated transition metals such as Cu(II), IMAGE was exploited to isolate several lipases from different sources and so evaluate their affinities for iminodiacetic acid (IDA)-Cu(II) and, in particular, to evaluate their enzymatic activities while retained on a gel (IMAGE) containing chelated copper [IDA-Cu(II)], It was found that all lipases can be active within the gel while being coordinated to IDA-Cu(II), It is concluded that active site histidine (most likely) is not involved in the metal recognition and thus other resident histidines serve as electron donors, This is mainly attributed to the known phenomenon of occlusion of the catalytic site by more or less rigid lid structures. The involvement of active site histidine in hydrogen bonding is also evoked.

关键词： catalytic triad immobilized metal-ion affinity gel electrophoresis interfacial activation lid lipases metal binding site structure-function relationships

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A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols

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PROTEIN SCIENCE 1996年第1期5卷 83-88页

作者： Holmquist, M Haeffner, F Norin, T Hult, K ROYAL INST TECHNOL DEPT CHEMS-10044 STOCKHOLMSWEDEN

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.

关键词： chiral recognition chiral resolution molecular modeling structure-function relationships substrate binding 2-methylalkanoic acid

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Protein function in the crystal

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ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR structure 1996年 25卷 343-365页

作者： Mozzarelli, A Rossi, GL Institute of Biochemical Sciences University of Parma Italy.

Protein crystals contain wide solvent-filled channels that allow for traffic of metabolites and intramolecular motility. Ligand binding, catalysis and allosteric regulation occur in the crystalline environment but intermolecular interactions may hinder function-associated transitions and alter activity with respect to solution. Lattice constraints have, however, provided the opportunity to isolate and characterize conformational states that are poorly populated in solution. New methods are being developed to initiate reactions rapidly and synchronously throughout the crystal and to monitor their time course. A model consistent with kinetics in the crystal is necessary to interpret the results of time-resolved macromolecular crystallography.

关键词： catalysis in enzyme crystals biological activity in the crystalline state structure-function relationships single-crystal microspectrophotometry polarized absorption spectroscopy X-ray crystallography of active intermediates

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On the functional significance of substrate accessibility in the photosynthetic water oxidation mechanism

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PHYSIOLOGIA PLANTARUM 1996年第2期96卷 342-350页

作者： Wydrzynski, T Hillier, W Messinger, J Research School of Biological Sciences The Australian National Univ. GPO Box 475 Canberra ACT 2601 Australia.

Recent evidence suggests that after selective perturbation of the protein structure in photosystem II, hydrogen peroxide is formed at the water oxidation catalytic site instead of molecular oxygen. In this communication, we review the interpretation of these observations and elaborate on the hypothesis that an essential factor in determining the end-product of photosynthetic water oxidation is one of substrate accessibility. It is argued that normally the access of water to the catalytic site is controlled by a hypdrophobic domain in the surrounding protein matrix and that the production of O-2 is optimized by an ordered binding of the two substrate water molecules. It is proposed that upon perturbation of the hydrophobic domain (for example, through the removal of various extrinsic proteins) the catalytic site becomes exposed to excess water from the external solvent phase. As a consequence, additional water binds at the catalytic site during intermediate oxidation steps and undergoes a partial oxidation reaction to form hydrogen peroxide. The importance of water accessibility to the structure/function relationships of photosystem II is discussed, particularly with respect to photoinhibitory damage through the formation of hydrogen peroxide.

关键词： hydrogen peroxide formation oxygen evolution photoinhibition photosystem II structure-function relationships

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