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A comprehensive, structural and functional, in silico analysis of the medium-chain dehydrogenase/reductase (MDR) superfamily, including 583 proteins, was carried out by use of extensive database mining and the BLASTP program in an iterative manner to identify all known members of the superfamily. Based on phylogenetic, sequence, and functional similarities, the protein members of the MDR superfamily were classified into three different taxonomic categories: (a) subfamilies, consisting of a closed group containing a set of ideally orthologous proteins that perform the same function; (b) families, each comprising a cluster of monophyletic subfamilies that possess significant sequence identity among them and might share or not common substrates or mechanisms of reaction; and (c) macrofamilies, each comprising a cluster of monophyletic protein families with protein members from the three domains of life, which includes at least one subfamily member that displays activity related to a very ancient metabolic pathway. In this context, a superfamily is a group of homologous protein families (and/or macrofamilies) with monophyletic origin that shares at least a barely detectable sequence similarity, but showing the same 3D fold.The MDR superfamily encloses three macrofamilies, with eight families and 49 subfamilies. These subfamilies exhibit great functional diversity including noncatalytic members with different subcellular, phylogenetic, and species distributions. This results from constant enzymogenesis and proteinogenesis within each kingdom, and highlights the huge plasticity that MDR superfamily members possess. Thus, through evolution a great number of taxa-specific new functions were acquired by MDRs. The generation of new functions fulfilled by proteins, can be considered as the essence of protein evolution. The mechanisms of protein evolution inside MDR are not constrained to conserve substrate specificity and/or chemistry of catalysis. In consequence, MDR functional diversity is more complex than sequence diversity.MDR is a very ancient protein superfamily that existed in the last universal common ancestor. It had at least two (and probably three) different ancestral activities related to formaldehyde metabolism and alcoholic fermentation. Eukaryotic members of this superfamily are more related to bacterial than to archaeal members; horizontal gene transfer among the domains of life appears to be a rare event in modern organisms.Keywords: protein taxonomy; protein evolution; mediumchain alcohol dehydrogenase; enoyl reductase; formaldehyde dehydrogenase.Correspondence to H. Riveros-Rosas, Depto. Bioquı´mica, Fac. Medicina, UNAM, Apdo. Postal 70-159, Cd. Universitaria, Me´xico, 04510, D.F., Me´xico. Fax: + 52 55 5616 2419, Tel.: + 52 55 5622 0829, E-mail: hriveros@servidor.unam.mx Abbreviations: AADH, allyl alcohol dehydrogenase; ACR, acyl-CoA reductase; ADH, alcohol dehydrogenase; AL, alginate lyase; ARP, auxinregulated protein; AST, membrane traffic protein; BCHC, 2-desacetyl-2-hydroxyethyl bacteriochlorophyll...

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Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.

Ustilago maydis mitochondria contain the four classical components of the electron transport chain (complexes I, II, III, and IV), a glycerol phosphate dehydrogenase, and two alternative elements: an external rotenone-insensitive flavone-sensitive NADH dehydrogenase (NDH-2) and an alternative oxidase (AOX). The external NDH-2 contributes as much as complex I to the NADH-dependent respiratory activity, and is not modulated by Ca2+, a regulatory mechanism described for plant NDH-2, and presumed to be a unique characteristic of the external isozyme. The AOX accounts for the 20% residual respiratory activity after inhibition of complex IV by cyanide. This residual activity depends on growth conditions, since cells grown in the presence of cyanide or antimycin A increase its proportion to about 75% of the uninhibited rate. The effect of AMP, pyruvate and DTT on AOX was studied. The activity of AOX in U. maydis cells was sensitive to AMP but not to pyruvate, which agrees with the regulatory characteristics of a fungal AOX. Interestingly, the presence of DTT during cell permeabilisation protected the enzyme against inactivation. The pathways of quinone reduction and quinol oxidation lack an additive behavior. This is consistent with the competition of the respiratory components of each pathway for the quinol/quinone pool.

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