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Abstract Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented.

Abstract Background Families of homologous enzymes evolved from common progenitors. The availability of multiple sequences representing each activity presents an opportunity for extracting information specifying the functionality of individual homologs. We present a straightforward method for the identification of residues likely to determine class specific functionality in which multiple sequence alignments are converted to an annotated graphical form by the Conserved Property Difference Locator (CPDL) program. Results Three test cases, each comprised of two groups of funtionally-distinct homologs, are presented. Of the test cases, one is a membrane and two are soluble enzyme families. The desaturase/hydroxylase data was used to design and test the CPDL algorithm because a comparative sequence approach had been successfully applied to manipulate the specificity of these enzymes. The other two cases, ATP/GTP cyclases, and MurD/MurE synthases were chosen because they are well characterized structurally and biochemically. For the desaturase/hydroxylase enzymes, the ATP/GTP cyclases and the MurD/MurE synthases, groups of 8 (of ~400), 4 (of ~150) and 10 (of >400) residues, respectively, of interest were identified that contain empirically defined specificity determining positions. Conclusion CPDL consistently identifies positions near enzyme active sites that include those predicted from structural and/or biochemical studies to be important for specificity and/or function. This suggests that CPDL will have broad utility for the identification of potential class determining residues based on multiple sequence analysis of groups of homologous proteins. Because the method is sequence, rather than structure, based it is equally well suited for designing structure-function experiments to investigate membrane and soluble proteins.

The germline chromosomes in Paramecium and other ciliated protozoa contain regions ()f DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5[prime]-TA-3[prime] dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5[prime]-TA-3[prime] and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) detective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5[prime]-TA-3[prime] dinucleotide. Failure to excise IES6649 also revealed a 29-bp lES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal lES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal lES is extremely rare or nonexistent. These results indicate that lESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an lES with its excision partner.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during' the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, lESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tc1 transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259l has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its wild-type micronuclear copy through multiple sexual generations.

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7°C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases. [ABSTRACT FROM PUBLISHER]