Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10% of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentosephosphate pathway

Aplicación de distintas metodologías a la monitorización de la humedad del suelo y al cálculo del balance hídrico diario

Dada la importancia de conocer la humedad del suelo de forma precisa y en tiempo real, se ha realizado este trabajo de investigación cuyo objetivo principal ha sido seleccionar un Balance Hídrico del Suelo (BHS) diario y validar sus estimaciones de humedad del suelo frente a medidas obtenidas “in situ”, aplicándolo a tres emplazamientos seleccionados en la zona centro con características edáficas y climáticas diferentes, y de este modo estimar con cierta precisión la humedad del suelo como Agua Disponible (AD) para las plantas y a su vez permitir la realización de estudios climáticos. Los observatorios meteorológicos seleccionados fueron: Guadalajara/El Serranillo en la zona aluvial del río Henares; Colmenar Viejo/Base Famet en la rampa sur del Guadarrama sobre rocas metamórficas; y Radiosondeo/Madrid(Barajas) en arenas arcósicas de grano grueso. Se realizó una caracterización morfológica y un estudio de las propiedades físicas, químicas e hidrofísicas de los suelos en cada emplazamiento. El suelo de Guadalajara, Xerorthent Típico presenta una secuencia genética de horizontes (Ap-AC-C1-C2) siendo su clase textural entre franco-arenosa a franca, con menos del 2% de elementos gruesos, presencia de caliza a lo largo de todo el perfil, destacando la homogeneidad en vertical y horizontal de sus propiedades. El suelo de Colmenar, Xerorthent Dystrico, presenta una secuencia genética de horizontes (A-C-C/R) apareciendo el horizonte C/R entre 20-30 cm; y la roca aproximadamente a unos 30 cm; destacando en este perfil su acidez y el alto contenido de elementos gruesos. El suelo de Radiosondeo, Haploxeralf Típico, presenta la secuencia normal de horizontes de los alfisoles (A-Bt1-Bt2-C/Bt); destacando su heterogeneidad principalmente en el plano horizontal, con presencia del Bt a diferentes profundidades en un corto espacio longitudinal. En una primera fase de experimentación (2007-2008) se seleccionaron BHS diarios que sólo utilizaban como datos de entrada la información de variables meteorológicas y el valor del Agua Disponible Total (ADT) para cada tipo de suelo y profundidad. Se probaron BHS diarios con agotamiento exponencial y directo de la reserva, utilizando la evapotranspiración de referencia de Penman-Monteith recomendada por FAO. Al mismo tiempo que se disponía de los datos estimados de humedad de suelo mediante diferentes BHS diarios en los tres emplazamientos, también se realizó una monitorización de la humedad del suelo “in situ” mediante el método gravimétrico, con adaptación de dicha metodología a la problemática de cada suelo, para determinar en cada fecha tanto la humedad del suelo como su contenido de AD para una profundidad de 0 a 30 cm. Se tomaron en cada fecha de muestreo 5 muestras para la profundidad 0- 10 cm, otras cinco para 10-20 cm y otras cinco para 20-30 cm, realizándose el correspondiente tratamiento estadístico de los datos. El ADT se calculó a partir de los datos de capacidad de campo y punto de marchitez obtenidos en laboratorio con membrana de Richards. Los resultados de esta primera fase permitieron conocer que el BHS exponencial diario era el que mejor estimaba el AD en Guadalajara considerando la capacidad de campo a una presión de 33 kPa, mientras que en Colmenar se debían considerar para un mejor ajuste, 10 kPa en lugar de 33 kPa. En el observatorio de Radiosondeo debido a que en cada fecha de muestreo la profundidad en la que aparecía el horizonte Bt era diferente, no se pudo demostrar si el BHS exponencial diario tenía un buen comportamiento. En una segunda fase de experimentación (2009-2012) y con el objeto de aminorar los problemas encontrados en Radiosondeo para la medida de humedad del suelo por el método gravimétrico, se procedió a la instalación y utilización de diferentes sensores de medida de humedad de suelo en el mismo observatorio: TDR (time domain reflectometry - TRIME T3 de IMKO); FDR capacitivo (frecuency domain reflectometry - ECH2O EC-20 de DECAGON) y otros. Esta segunda fase de experimentación tuvo una duración de 4 años y se compararon las medidas de humedad de suelo obtenidas a partir de los sensores con las estimadas del BHS exponencial hasta una profundidad de 0 a 85 cm. En laboratorio se realizaron calibraciones específicas de los sensores TDR y FDR para cada uno de los horizontes más diferenciados del Haploxeralf Típico, utilizando diferentes tipos de regresión. Los valores de humedad de suelo con el equipo TDR, corregidos mediante la calibración específica de laboratorio, fueron los que más se ajustaron a las medidas realizadas por método gravimétrico “in situ”, por lo que se utilizó el TDR para las comparaciones con los valores obtenidos del BHS exponencial diario durante los cuatro años de esta segunda fase experimental. Se realizaron diferentes estimaciones del ADT, partiendo de datos de laboratorio y/o de datos procedentes de humedad de los sensores en campo. Los resultados mostraron de nuevo la conveniencia de utilizar el BHS exponencial diario, pero en este caso, con la estimación del ADT realizada a partir de las gráficas de los sensores. Mediante la utilización de los datos de humedad del BHS exponencial diario se han realizado comparaciones con el mismo tipo de balance pero utilizando un periodo semanal o mensual en lugar de diario, para conocer las diferencias. Los valores obtenidos con periodicidad mensual han dado valores de AD inferiores a los balances calculados semanalmente o diariamente. Por último se ha comprobado que los resultados de un BHS exponencial diario pueden complementar la información que se obtiene del Índice de Precipitación Estandarizado (SPI) y pueden mejorar el estudio de la sequía agrícola. ABSTRACT Due to the importance of a better knowledge of soil water at real time and in a more precisely way, this research work has being carried out with the main objective of selecting a daily Soil Water Balance (SWB) to estimate soil water content, and validate it in comparison to “in situ” measurements. Three locations, differing in soil and climate characteristics, were selected in central Spain in order to estimate with certain acuity soil water as plant-Available Water (AW) and to serve as a tool for the climatic studies. The selected places near meteorology stations were: Guadalajara/El Serranillo an alluvium of the Henares watershed; Colmenar Viejo/Base Famet, in the south raised area of the Guadarrama river basin, over metamorphic rocks; and Radiosondeo/Madrid (Barajas) in coarse arkosic sandstone. Morphology characterization, physical, chemical and hydrologic soil properties were studied in each area. In Guadalajara the soil is a Typic Xerorthent with a (Ap-AC-C1- C2) genetic horizon sequence, loam-sandy to loam textural class, less than 2% of rock fragments, presence of equivalent CaCO3 through the whole profile, outstanding the vertical and horizontal homogeneity of the properties. In Colmenar the soil is represented by a Dystric Xerorthent with a (A-C-C/R) genetic horizon sequence, the C/R is 20-30 cm deep where rock outcrops are approximately at 30 cm; a characteristic feature of this profile is its high acidity and high rock fragments content. In Radiosondeo the soil is represented by a Typic Haploxeralf, with the usual alfisol genetic horizon sequence (A-Bt1-Bt2-C/Bt); outstanding its horizontal heterogeneity, “the variability of the Bt (clay enriched horizon) depth in short distances”. In a first experimental stage (2007-2008), the daily SWB chosen was that which only uses as input data the information from the meteorology variables and plant-Total Available Water (TAW) for each soil type and depth. Different daily SWB (with exponential or direct plant-Available Water depletion) were applied, using the Penman- Monteith reference evapotranspiration (ETo) recommended by FAO. At the same time as soil water content was estimated from the different daily SWB at the three locations, also soil water content was being monitored by “in situ” gravimetric methodology, adapting it to each soil characteristic, to determine every time soil water content and AW to a depth of 0 to 30 cm. In each sampling date, 5 samples for each depth were taken: 0-10 cm; 10-20 cm and 20-30 cm and the data were submitted to the corresponding statistical analysis. The TAW was calculated based on field capacity (FC) and permanent wilting point (PWP) data obtained from laboratory by the Richards pressure plate. Results from this first experimental stage show that the daily exponential SWB was the one which better estimated the AW in Guadalajara considering field capacity at -33 kPa, though in Colmenar, field capacity at -10 kPa must be considered instead of -33 kPa for a better estimation. In Radiosondeo due to the fact that the Bt horizon depth varied in different sampling dates, it could not be established if the daily exponential SWB had a good performance. In a second experimental stage (20019-2012) and with the objective of minimizing the problems encountered in Radiosondeo for measuring “in situ” soil water content by the gravimetric method, the installation of different sensors for measuring soil water content were established and used in the same field location: TDR (time domain reflectometry - TRIME T3 from IMKO), capacitance FDR (frecuency domain reflectometry - ECH2O EC-20 from DECAGON) and others. This second experimental stage lasted 4 years in order to compare the soil water measures from the sensors with the estimations by the exponential SWB form 0 to 85 cm soil depths. At laboratory, specific calibrations for TDR and FDR sensors of the Typic Haploxeralf more differentiated horizons were done using different types of regressions. The results showed that soil water data obtained by the TDR equipment, corrected by the specific laboratory calibration, best fitted to “in situ” gravimetric soil water measures. In this way TDR was used for comparing to the daily exponential SWB during the four years of this second experimentation stage. Various estimations for obtaining TAW were tested; based on laboratory data – and/or on the data obtained of the soil water content field sensors. Results confirmed again, the convenience of using the daily exponential SWB, though in this case, with the TAW obtained from the field sensors graphics. Soil water estimated by exponential SWB on daily basis was compared to weekly and monthly periods, in order to know their reliability. The results obtained for a monthly period gave less AW than the ones obtained in a weekly or daily period. Finally it has been proved that the results obtained from the exponential SWB in a daily bases can be used as a useful tool in order to give complementary information to the SPI (Precipitation Standardized Index) and to help in agricultural drought studies.

Palaeoenvironmental reconstruction of Las Conchas Mire (Asturias, N. Spain) from the ketone and fatty acid record= reconstrucción paleoambiental de la Turbera de las Conchas (Asturias, N. España)a partir del registro de cetonas y ácidos grasos

Se han estudiado los biomarcadores, principalmente cetonas y ácidos, preservados en el registro de 3.2 m de la Turbera de Las Conchas. Las cetonas reflejan cierta actividad bacteriana desde 94 cm hasta la base del registro, Los ácidos grasos reflejan una buena preservación de la materia orgánica, salvo en los 20 cm superiores en los que hay indicios de oxidación microbiana de alcanos .The biomarkers, mainly ketones and fally aclds, preserved In 3.2 m deep Las Conchas Mire have been studied, Kelones reflect certain bacterial activity from 94 cm to the bottom of the record. Falty aclds indlcate a good preservation of the organlc matter, wlth the exception of the uppermost 20 cm In whlch mlcroblal oxldation of alkanes are likely to occur

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

An amino acid as a cofactor for a catalytic polynucleotide

Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the in vitro selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires l-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of ≈1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA–histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the “RNA world” may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.

Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch

Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base. The biological outcome is the prevention of C/G→A/T transversions. The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood. In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex. After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity. The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA. It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh–DNA complex. This stimulation is independent of the catalytic activity of Ape1. Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency. Our study suggests that protein–protein interactions may occur in vivo to achieve efficient BER of A/GO.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492–742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching

The nucleocapsid protein (NC) of HIV type 1 is a nucleic acid chaperone that facilitates the rearrangement of nucleic acids into conformations containing the maximum number of complementary base pairs. We use an optical tweezers instrument to stretch single DNA molecules from the helix to coil state at room temperature in the presence of NC and a mutant form (SSHS NC) that lacks the two zinc finger structures present in NC. Although both NC and SSHS NC facilitate annealing of complementary strands through electrostatic attraction, only NC destabilizes the helical form of DNA and reduces the cooperativity of the helix-coil transition. In particular, we find that the helix-coil transition free energy at room temperature is significantly reduced in the presence of NC. Thus, upon NC binding, it is likely that thermodynamic fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations to find the lowest energy state. The reduced cooperativity allows these fluctuations to occur in the middle of complex double-stranded structures. The reduced stability and cooperativity, coupled with the electrostatic attraction generated by the high charge density of NC, is responsible for the nucleic acid chaperone activity of this protein.

Abscisic Acid-Dependent and -Independent Expression of the Carrot Late-Embryogenesis-Abundant-Class Gene Dc3 in Transgenic Tobacco Seedlings1

We studied the expression of three promoter 5′ deletion constructs (−218, −599, and −1312) of the LEA (late embryogenesis abundant)-class gene Dc3 fused to β-glucuronidase (GUS), where each construct value refers to the number of base pairs upstream of the transcription start site at which the deletion occurred. The Dc3 gene is noted for its induction by abscisic acid (ABA), but its response to other plant hormones and various environmental stresses has not been reported previously for vegetative cells. Fourteen-day-old transgenic tobacco (Nicotiana tabacum L.) seedlings were exposed to dehydration, hypoxia, salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS activity was quantified fluorimetrically and expression was observed by histochemical staining of the seedlings. An increase in GUS activity was observed in plants with constructs −599 and −1312 in response to dehydration and salinity within 6 h of stress, and at 12 h in response to hypoxia. No increase in endogenous ABA was found in any of the three lines, even after 72 h of hypoxia. An ABA-independent increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 μL L−1 ethylene in air or 100 μm MeJa. Virtually no expression was observed in construct −218 in response to dehydration, salinity, or MeJa, but there was a moderate response to ethylene and hypoxia. This suggests that the region between −218 and −599 is necessary for ABA (dehydration and salinity)- and MeJa-dependent expression, whereas ethylene-mediated expression does not require this region of the promoter.

Epicuticular Wax Accumulation and Fatty Acid Elongation Activities Are Induced during Leaf Development of Leeks1

Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0- and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.

Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin.

The application of an external electric field to dry films of Asp-85-->Asn mutant bacteriorhodopsin causes deprotonation of the Schiff base, resulting in a shift of the optical absorption maximum from 600 nm to 400 nm. This is in marked contrast to the case of wild-type bacteriorhodopsin films, in which electric fields produce a red-shifted product whose optical properties are similar to those of the acid-blue form of the protein. This difference is due to the much weaker binding of the Schiff-base proton in the mutant protein, as indicated by its low pK of approximately 9, as compared with the value pK approximately 13 in the wild type. Other bacteriorhodopsins with lowered Schiff-base pK values should also exhibit a field-induced shift in the protonation equilibrium of the Schiff base. We propose mechanisms to account for these observations.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.