Reviews

Book Reviewed in this article:
La Symbiose Mycorhizienne. Ed. by J. A. FORTIN, C. CHAREST and Y. PICHÉ.
Lichen Biology. Ed. by T homas H. NASH III.
The Natural History of Pollination. By MICHAEL PROCTOR, PETER YEO and ANDREW LACK.
Flora of the Venezuelan Guayana, vol. 2, Pteridophytes, Spermatophytes: Acanthaceae-Araceae. Ed. by JULIAN A. STEYERMARK, PAUL E. BERRY and BRUCE K. HOLST.     

Structural, biochemical, and phylogenetic analyses suggest that indole-3-acetic acid methyltransferase is an evolutionarily ancient member of the SABATH family

The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 A resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants.     

Reviews

book reviewed in this article
The Population Structure of Vegetation , ( Handbook of Vegetation Science 3. ) Ed. by J. WHITE.
An Introduction to Plant Cell Development. By J. BURGESS.
Hormone Binding Sites in Plants. By M ichael V enis .
Seed Ecology. By MICHAEL, FENNER.
Inorganic Carbon Uptake by Aquatic Photosynthetic Organisms. Ed. by William J. Lucas and Joseph A. Berry.     

Evolution of Cinnamate/p-coumarate carboxyl methyltransferases and their role in the biosynthesis of methylcinnamate

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.     

REVIEWS

Books Review in this article:
Forest Trees and Timbers of the British Empire. Part I. Some East African Coniferae and Leguminosae. By L. C halk , M.A., D.Phil., J. B urtt D avy , M.A., Ph.D. and H. E. D esch , B.SC.
Part II. Twenty West African Timber Trees. By L. C halk , M.A., D.Phil., J. B urtt D avy , M.A., Ph.D., H. E. D esch , B.SC, M.A. and A. C. H oyle , B.Sc, M.A.
Part III. Fifteen South African High Forest Timber Trees. By L. C halk , M.A., D.Phil., M. M. C hattaway , B.SC, M.A., J. B urtt D avy , M.A., Ph.D., F. S. L aughton , B.SC. and M. H. S cott , B.SC.
Colloids in Agriculture. By C. E. M arshall .
Flore Laurentienne. By F rère M arie -V ictorin , D.SC.
Plant Viruses. By K enneth M. S mith .
Plant Life: A Text-book of Botany. By D. B. S wingle .
Gardening in East Africa: A Practical Handbook. Edited by A. J. J ex -B lake , with a foreword by Sir A rthur W. H ill .
British Stem- and Leaf-Fungi (Coelomycetes). Vol. I. Sphaeropsidales. By W. B. G rove , M.A.     

Identification of SABATH Family Members in Solanum lycopersicum and Their Expression Patterns Under Abiotic/Biotic Stresses

Plant Molecular Biology Reporter - The SABATH family proteins catalyse S-adenosyl-l-methionine-dependent methylation of hormones, signal molecules and other metabolites in plants, and they play an...     

Book Reviews

EFFECTS OF LOW TEMPERATURES ON BIOLOGICAL MEMBRANES . Edited by G. J. Morris & A. Clark
CELL DIFFERENTIATION: MOLECULAR BASIS AND PROBLEMS . Edited by L.Edited by L. NOVER, M.LUCKNER & B. PARTHER
ECOLOGICAL BULLETINS 30. ECOLOGY OF A SUBARCTIC MIRE . Edited by M. SVENSSON
ENCYCLOPEDIA OF PLANT PHYSIOLOGY, VOLUME 13A. PLANT CARBOHYDRATES I. INTRACELLULAR CARBOHYDRATES . Edited by F. A. LOEWUS & W. TANNER
COMPONENTS OF PRODUCTIVITY OF MEDITERRANEAN-CLIMATE REGIONS. BASIC AND APPLIED ASPECTS . Edited by N. S. M arganis & H. A. M^OONEY
ENCYCLOPEDIA OF PLANT PHYSIOLOGY, VOLUME 13B. PLANT CARBOHYDRATES H. EXTRACELLULAR CARBOHYDRATES . Edited by W. T anner & F. A. L oewus
PHOTOSYNTHESIS BIBLIOGRAPHY, VOLUME 8 1977, VOLUME 9 1978, VOLUME 101 1979 . Edited by Z. SESTAK & J. CATSKY
THE CHANGING CLIMATE: RESPONSES OF THE NATURAL FLORA AND FAUNA. By MICHAEL J. FORD     

Reviews

Advances in Botanical Research . Vol. 14. Ed. by J. A. C allow .
Inorganic Nitrogen Metabolism . Ed. by W. R. U llrich , P. J. A paricio , P. J. S yrett and F. C astillo .
A Centry od Nitrogen Fixation Research . Ed. by F. J. B ergersen and J. R. P ostgate .
Horizons in Lichenology . Ed. by D. H. D alby , D. L. H awksworth and S. L. J ury .
Introduction to Ecological Biochemistry , By J. B. H arborne .
Russell's Soil Conditions and Plant Growth, Ed. by A lan W ild .
Disease and Plant Population Biology . By J. J. B urdon.
Molecular Determinants of Plant Diseases . Ed. by S. N ishimura , C. P. V ance and N. D oke.
British Fungus Flora, Agrics and Boleti 5. Strophariaceae & Coprinaceae P.P.: Hypholoma, Melanotus, Psilocybe, Stropharia, Lacrymaria & Panaeolus, Psilocybe, Stropharia, Lacrymaria & Panaeolus, By R W atling and M. N. G regory .
Mechanisms of Woody Plant Defenses Against Insects . Ed. by W. J. M attson , J. L evieux and C. B ernard -D agan .
Air Pollution and Acid Rain. The Biological Impact . By A. W ellburn .
The Australian Paniceae (Paniceae) . By R. D. W erster .
Kew Index for 1986 . Compiled by R.A. D avies and K.M. L loyd .
Kew Index for 1987 . Compiled by R. A. D avies and K. M. L loyd .
Index Kewensis Supplement XVII . Compiled by J. L. M. P inner , T. A. B ence , R. A. D avies and K. M. L loyd .
Index Kewensis Supplement XVIII . Compiled by J. L. M. P inner , T. A. B ence , R. A. D avies and K. M. L loyd . Edited by R. A. D avies .
The Botany of Mangroves . By P. B. T omlinson .
Floweroing Plants in West Africa . By M argaret S teentoft .
Plants in Danger . By S. D. D avies     

Book Reviews

CLONING AGRICULTURAL PLANTS VIA IN VITRO TECHNIQUES . Edited by B. V. C onger
EFFECTS OF HEAVY METAL POLLUTION ON PLANTS. VOLUME 1. EFFECTS OF TRACE METALS ON PLANT FUNCTION (352 pp.). VOLUME 2. METALS IN THE ENVIRONMENT (257 pp.). Edited by N. W. L epp
STOMATAL PHYSIOLOGY . Edited by P. G. J arvis AND T. A. M ansfield
DEVELOPMENTS IN PLANT AND SOIL SCIENCES. VOLUME 3. CARBON AND NITROGEN METABOLISM . Edited by J. D. B ewley
ECOLOGICAL STUDIES. VOLUME 36. PHYSIOLOGICAL PROCESSES IN PLANT ECOLOGY. TOWARD A SYNTHESIS WITH ATRIPLEX , By C. B. O smond , O. B jörkman and D. J. A nderson
PHYSIOLOGICAL PROCESSES LIMITING PLANT PRODUCTIVITY . Edited By C. B. J ohnson
MATHEMATICS AND PLANT PHYSIOLOGY . Edited by D. A. R ose and D. A. C harles -E dwards
BIOSYNTHESIS OF ISOPRENOID COMPOUNDS, VOLUME 1 . Edited by J. W. P ortkr and S. L. S purgeon
STRUCTURE AND FUNCTION OF PLANT ROOTS . Edited by R. B rouwhr , O. G apaiuková , J. K olek & B. C. L oughman
THE CELL CYCLE (Society for Experimental Biology, Seminar Series No. 10). Edited by P. C. L. J ohn     

A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the “tip of the iceberg” since thousands of sequences remain uncharacterized in this enzyme family alone.     

Book Reviews

Books reviewed in this article:
W orld F ederation for C ulture C ollections T eaching V ideo (1986).
A C olour A tlas of M icrobiology (1986). R. J. Olds.
A C olour A tlas of I nfectious D iseases 2nd edition (1987). R. T. D. Emond & H. A. K. Rowland.
D iagnostic P icture T ests in I nfectious D iseases (1987). R. T. D. Emond & H. A. K. Rowland
T he A ntimicrobial A gents A nnual (1987). Edited by P. K. Peterson & J. Verhoef.
T he B acteria. Volume 10. T he B iology of Pseudomonas (1986). Edited by J. R. Sokatch.
T he I nfluence of A ntibiotics on the H ost -P arasite R elationship II (1985). Edited by D. Adam, H. Hahn & W. Opperkuch.     

Reviews

The Flavonoids. Advances in Research since 1980. Ed. by J. B. H arborne .
Hungry crops : a guide to nutrient deficiencies in field crops By N. J. G rundon .
Microbial ecology: organisms, habitats, activities. By H einz S tolp .
Evolutionary Biology of the Fungi. Ed. by A. D. M. R ayner , C. M. B rasier and D. M oore .
Micro-algal Biotechnology . Edited by M.A. B orowitzka and L. J. B orowitzka and L. J. B orowitzka .
Experimental Phycology: A laboratory manual Edited by C hristopher S. L obban , D avid J. C hapman , and B runo P. K remer .
100 Families of Flowering Plants. By M. H ickey and C. K ing .
Differentiation Patterns in Higher Plants. Edited by K. M. U rbanska .
The Living Tundra. By Yu. I. C hernov , Translated by D. L öve .
North American Terrestrial Vegetation. Edited by M. G. B arbour & W. D. B illings .
Biogeography and Quaternary History in Tropical Latin America. By T. C. W hitmore and G. T. P rance (Editors).
Biogeographical Evolution of the Malay Archipelago . By T. C. W hitmore (Editor).
Postglacial Vegetation of Canada. By J. C. R ichie .     

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).     

The phylogenetic problem of Huia (Amphibia: Ranidae)

A taxonomic consensus for the diverse and pan-global frog family Ranidae is lacking. A recently proposed classification of living amphibians [Frost, D.R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006. The amphibian tree of life. B. Am. Mus. Nat. Hist. 297, 1-370] included expansion of the Southeast Asian ranid frog genus Huia from seven to 47 species, but without having studied the type species of Huia. This study tested the monophyly of this concept of Huia by sampling the type species and putative members of Huia. Molecular phylogenetic analyses consistently recovered the type species H. cavitympanum as the sister taxon to other Bornean-endemic species in the genus Meristogenys, rendering all previously published concepts of Huia as polyphyletic. Members of Huia sensu [Frost, D.R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006. The amphibian tree of life. B. Am. Mus. Nat. Hist. 297, 1-370.] appear in four places within the family Ranidae. A clade containing the type species of Odorrana is phylogenetically unrelated to the type species of Huia, and Odorrana is removed from synonymy with Huia. These findings underscore the need to include relevant type species in phylogenetic studies before proposing sweeping taxonomic changes. The molecular phylogenetic analyses revealed a high degree of homoplasy in larval and adult morphology of Asian ranid frogs. Detailed studies are needed to identify morphological synapomorphies that unite members in these major clades of ranid frogs.     

REVIEWS

Book review in this article
Botanical Latin . By W. T. S tearn .
Laboratory Manual of Tropical Mycology and Elementary Bacteriology . By A. T hompson and G loria L im .
Agriculture in the Tropics . By C. C. W ebster and P. N. W ilson .
Bananas . By N. W. S immonds .
Rice . By D. H. G rist .
Tea . By T. E den .
Lowson's Botany . By E. W. S imon , K. J. D ormer and J. N. H artshorne .
Annual Review of Plant Physiology , Vol. 16. Ed. by L. M achlis and W. K. B riggs .
Ultrastructural Plant Cytology . By A. F rey -W yssling and K. M ühlethaler .
Drawings of British Platits . Part XXII, Scrophulariaceae (1). By S tella R oss
Molecules and Cells .By D. A. C oult . The Principles of Modern Biology. Ed. by D. A. C oult .
The Growth of Cereals and Grasses . Ed. by F. L. M ilthorpe and J. D. I vins .
Origins of Metidelism . By R. C. O lby .
Fungal Genetics . By J. R. S. F incham and P. R. D ay . Botanical Monographs, Vol. 4, ed. by W. O. J ames .
Genetik der Pilze . by K. E sser and R. K uenen .
Microbial and Molecular Genetics . By J. R. S. F incham . Modern Biology Series, ed. by J. E. W ebb .
Index Kewensis Plantarum Phanerogamarum, Supplementum XIII . Compiled at the Herbarium of the Royal Botanic Gardens, Kew, under the direction of Sir G eorge T aylor .
A Flora of Northeastern Minnesota . By O lga L akela .
Stephen Hales, D.D., F.R.S. An Eighteenth Century Biography . By A. E. C lark -K ennedy .     

BOOK REVIEWS

Book reviewed in this article:
Central Pennsylvania Halloween Customs . Scientific direction by MAURICE A. MOOK, produced by D. P. DUVALL, L. P. GREENHILL, and C. CONKLIN
L'Aubrac filmed by JEAN-DOMINIQUE LAJOUX
L'Homme des Burons , filmed by J. D. LAJOUX
Le Forgeron des Hermaux , filmed by J. D. LAJOUX
Fléaux en cadence , filmed by J. D. LAJOUX
Foires d'Aubrac ou le Langage des Gestes , filmed by J. D. LAJOUX
Le Joug filmed by J. D. LAJOUX     

Trimethoprim binds in a bacterial mode to the wild-type and E30D mutant of mouse dihydrofolate reductase

Previous crystallographic studies of the antibacterial trimethoprim in complexes with bacterial and avian dihydrofolate reductases have shown substantial differences in the mode of binding, providing plausible explanations for the origin of the remarkable species selectivity of this inhibitor (Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., Kaufman, B. T., Beddell, C. R., Champness, J. N., Stammers, D. K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391; Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., and Kraut, J. (1985) J. Biol. Chem. 260, 392-399). A major species difference between the active sites is that the only carboxylate present is always Glu in vertebrates and Asp in bacteria. Crystallographic studies of the wild-type and E30D mutant of the enzyme from mouse now reveal that in both cases trimethoprim is bound in an identical fashion to that observed with the bacterial enzyme, and there is no obvious single explanation for the origin of the 10(5)-fold selectivity of trimethoprim binding. In an earlier study of a mouse wild-type enzyme using more limited data it was proposed that trimethoprim bound in the avian mode (Stammers, D. K., Champness, J. N., Beddell, C. R., Dann, J. G., Eliopoulos, E. E., Geddes, A. J., Ogg, D., and North, A. C. T. (1987) FEBS Lett. 218, 178-184), but a re-examination indicates that the occupancy of the active site by trimethoprim is less than had been thought, and we are currently unable to make an unambiguous interpretation of the electron density maps and cannot confirm the avian mode of binding in those crystals.     

REVIEWS

Grazing in Terrestrial and Marine Environments (British Ecological Society Symposium No. 4). Edited by D. J. C risp
Fundamentals of Botany Series . Edited by W. A. J ensen and L. G. K avaljian
Multivariate Statistical Analysis for Biologists . By H ilary S eal
Differenciation des Cellules Sexuelles et Eécondation chez les Cryptogames (Protoplasmatologia , Vol. VII/3b). By B. V azart
Allgemeine Botanik . By W. N ultsch
Piante Medicinali, Chimica, Farmacologia e Terapia . Vol. II. By R. B enigni , C. C apra and P. E. C attorini
Thermophilic Fungi . By D onald G. C ooney and R alph E merson
The Genera of Flowering Flants (Angiospermae). Based Principally on the Genera Plantarum of G. Bentham and J. D. Hooker. Dicotyledones . vol. 1. By J. H utchinson
Algal Cultures and Phytoplankton Ecology . By G. E. F ogg
Flora Europaea, Vol . 1. Eycopodiaceae to Plantanaceae . Edited by T. G. T utin , V. H. H eywood , N. A. B urges , D. H. V alentine , S. M. W alters and D. A. W ebb
The Biology of the Algae . By F. E. R ound
Flora of the British Isles Illustrations . Part 4. Monocotyledons . Drawings by S ybil J. R oles
Plants and the Ecosystem . By W. D. B illings . (Fundamentals of Botany Series, edited by W. A. J ensen and L. G. K avaljian
Essays on Crop Evolution . Edited by S ir J oseph H utchinson     

REVIEWS

Books reviewed in this article:
Plant Anatomy: Experiment and Interpretation , Part II, Organs. By E lizabeth G. C utter .
Genetics. By D. J. C ove .
The Structure and Life of Bryophytes. By E. V. W atson .
Underwater Science. Ed. by J. D. W oods and J. J. L ythgoe .
8ème Symposium Européen sur les Maladies à Virus des Arbres Fruitiers.
Evolution and Plants of the Past. By H. P. B anks .
Project Aqua. A Source Book of Inland Waters Proposed for Conservation. IBP Handbook No. 21. By H. L uther and J. R zóska .
Variation and Adaptation in Plant Species. By D. A. J ones and D. A. W ilkins .
Flora of Lesotho (Basutoland). By A my J acot G uillarmod .
Introduction to Biogeography. By B rian S eddon .
Trees: Structure and Function. By M. H. Z immerman and C. L. B rown ;
Flora of New Zealand , Volume II. By L. B. M oore and E. E dgar .     

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.