Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain.

Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.

Analysis of two incompletely spliced Arabidopsis cDNAs encoding novel types of peroxidase.

Most known class III peroxidase genes contain three introns at conserved positions. Two Arabidopsis cDNAs (ESTs), encoding novel type peroxidases ATP9a and ATP15a were sequenced, and found to contain inserts for intron 2. PCR and sequence analysis of genomic DNA revealed that the atp9a gene contains all three introns, whereas atp15a contains only introns 2 and 3. The ATP15a cDNA intron contained a single base substitution reducing the splicing potential significantly as compared with the genomic sequence. The putative enzymes share essential catalytic and structural features with horseradish peroxidase, despite a pair-wise sequence identity of only 40-45% among the three.

Widespread occurrence of a highly conserved RING-H2 zinc finger motif in the model plant Arabidopsis thaliana.

Several novel Arabidopsis thaliana proteins containing a RING-H2 zinc finger motif were predicted after database searches. Alignment of 29 RING-H2 finger sequences shows that the motif is strikingly conserved in otherwise unrelated proteins. Only short, non-conserved polar/charged sequences distinguish these domains. The RING-H2 domain is most often present in multi-domain structures, a number of which are likely to contain a membrane-spanning region or an additional zinc finger. However, there are several small (126-200 residues) proteins consisting of an N-terminal domain, rich in aliphatic residues, and a C-terminal RING-H2 domain. Reverse-transcription PCR suggests that the RING-H2 genes are widely expressed at low levels.

Computational analyses and annotations of the Arabidopsis peroxidase gene family.

Classical heme-containing plant peroxidases have been ascribed a wide variety of functional roles related to development, defense, lignification, and hormonal signaling. More than 40 peroxidase genes are now known in Arabidopsis thaliana for which functional association is complicated by a general lack of peroxidase substrate specificity. Computational analysis was performed on 30 near full-length Arabidopsis peroxidase cDNAs for annotation of start codons and signal peptide cleavage sites. A compositional analysis revealed that 23 of the 30 peroxidase cDNAs have 5' untranslated regions containing 40-71% adenine, a rare feature observed also in cDNAs which predominantly encode stress-induced proteins, and which may indicate translational regulation.

From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase.

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.

Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control. Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture. In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds. The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.

A circularly permuted alpha-amylase-type alpha/beta-barrel structure in glucan-synthesizing glucosyltransferases.

A motif of amino acid residues, located at the active site and specific beta-strands in alpha-amylases, is recognized in alpha-1,3- and alpha-1,6-glucan-synthesizing glucosyltransferases, leading to the conclusion that these enzymes contain an alpha/beta-barrel closely related to the (beta/alpha)8-fold of the alpha-amylase superfamily. The secondary structure elements of the transferase barrel, however, are circularly permuted to start with an alpha-helix equivalent to helix 3 in the alpha-amylases. Thus, the transferase counterpart of the long third beta-->alpha connection--constituting a domain in the alpha-amylases--is divided to precede and succeed the barrel. This architectural arrangement may be coupled to sucrose scission and glucosyl transfer. The involvement in the mechanism in glucosyltransferases of active site residues recurring in amylolytic enzymes is discussed.

Starch- and glycogen-debranching and branching enzymes: prediction of structural features of the catalytic (beta/alpha)8-barrel domain and evolutionary relationship to other amylolytic enzymes.

Sequence alignment and structure prediction are used to locate catalytic alpha-amylase-type (beta/alpha)8-barrel domains and the positions of their beta-strands and alpha-helices in isoamylase, pullulanase, neopullulanase, alpha-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes--all enzymes involved in hydrolysis or synthesis of alpha-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of beta-->alpha loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved beta-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.

Comparison of the domain-level organization of starch hydrolases and related enzymes.

Structure-prediction and hydrophobic-cluster analysis of several starch hydrolases and related enzymes indicated the organization of eleven domain types. Most enzymes possess a catalytic (beta/alpha)8-barrel and a smaller C-terminal domain as seen in crystal structures of alpha-amylase and cyclodextrin glucanotransferase. Some also have a starch-granule-binding domain. Enzymes breaking or forming endo-alpha-1,6 linkages contain domains N-terminal to the (beta/alpha)8-barrel.

Amino acid sequences and structures of chicken and turkey beta 2-microglobulin.

The complete amino acid sequences of chicken and turkey beta 2-microglobulins have been determined by analyses of tryptic, V8-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta 2-microglobulin supports the sequence-derived Mr of 11,048. The higher apparent Mr obtained for the avian beta 2-microglobulins as compared to human beta 2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta 2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta 2-microglobulin at 46 and 47 positions, respectively, and to bovine beta 2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta 2-microglobulins. The known X-ray crystallographic structures of bovine beta 2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of beta 2-microglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta 2-microglobulin can substitute the chicken beta 2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.