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Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 10 2 M -1 s -1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects k cat , by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Objectives: To characterise the serological and clinical findings in primary Sjögren's syndrome in which anti-La was found without anti-Ro. We hypothesised that a significant portion of these are falsely negative for anti-Ro60., Methods: Twenty-nine sera from primary Sjögren's syndrome patients were tested for antibodies directed against La and Ro. Anti-La was detected using bovine La treated with or without DNAase and RNAase to identify potential false positivity. Anti-Ro60 antibodies were detected using HEp-2000 substrate (in which cells are transfected with human Ro60) and HEp-2 substrate. Anti-Ro60 and Ro-52 were also tested by in vitro transcription/translation followed by immunoprecipitation assay., Results: All 29 sera bound La, even after treatment with DNAase and RNAase. Of the 29 sera, 25 were unequivocally negative on HEp-2000 (1:40 dilution). Four samples were anti-Ro60 positive with a speckled pattern, three of the four at 1:320 dilution. Thus, false negative anti-Ro60 exists in a small fraction (14%) of the Ro-negative/La-positive primary Sjögren's patients. However, all the samples were negative for Ro60 and Ro52 by in vitro immunoprecipitation assay. Clinically these patients tended not to have salivary gland pathology characteristic of Sjögren's syndrome., Conclusions: We found only a small fraction of Ro negative/La positive sera to show positive HEp-2000 pattern. These subjects did not have characteristic findings on pathological examination of minor salivary glands, suggesting these subjects have a process distinct from Sjögren's syndrome.