Keith A. Kretz

The hyperthermophile Nanoarchaeum equitans is an obligate symbiont growing in coculture with the crenarchaeon Ignicoccus. Ribosomal protein and rRNA-based phylogenies place its branching point early in the archaeal lineage, representing the new archaeal kingdom Nanoarchaeota. The N. equitans genome (490,885 base pairs) encodes the machinery for information processing and repair, but lacks genes for lipid, cofactor, amino acid, or nucleotide biosyntheses. It is the smallest microbial genome sequenced to date, and also one of the most compact, with 95% of the DNA predicted to encode proteins or stable RNAs. Its limited biosynthetic and catabolic capacity indicates that N. equitans' symbiotic relationship to Ignicoccus is parasitic, making it the only known archaeal parasite. Unlike the small genomes of bacterial parasites that are undergoing reductive evolution, N. equitans has few pseudogenes or extensive regions of noncoding DNA. This organism represents a basal archaeal lineage and has a highly reduced genome.

Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.

Several complementary DNAs (cDNAs) coding for sphingolipid activator protein-2 (SAP-2) were isolated from a lambda gt-11 human hepatoma library by means of polyclonal antibodies. The nucleotide sequence of the largest cDNA was colinear with the derived amino acid sequence of SAP-2 and with the nucleotide sequence of the cDNA coding for the 70-kilodalton precursor of SAP-1 (SAP precursor cDNA). The coding sequence for mature SAP-2 was located 3' to that coding for SAP-1 in the SAP precursor cDNA. Both SAP-1 and SAP-2 appeared to be derived by proteolytic processing from a common precursor that is coded by a genetic locus on human chromosome 10. Two other domains similar to SAP-1 and SAP-2 were also identified in SAP precursor protein. Each of the four domains was approximately 80 amino acid residues long, had nearly identical placement of cysteine residues, potential glycosylation sites, and proline residues. Each domain also contained internal amino acid sequences capable of forming amphipathic helices separated by helix breakers to give a cylindrical hydrophobic domain that is probably stabilized by disulfide bridges. Protein immunoblotting experiments indicated that SAP precursor protein (70 kilodaltons) as well as immunoreactive SAP-like proteins of intermediate sizes (65, 50, and 31 kilodaltons) are present in most human tissues.

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62 degrees C for 1 h and 27% of its initial activity after 10 min at 85 degrees C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

Saposin A, a heat-stable 16-kDa glycoprotein, was isolated from Gaucher disease spleen and purified to homogeneity. Chemical sequencing from its amino terminus and of peptides obtained by digestion with protease from Staphylococcus aureus strain V-8 demonstrated that saposin A is derived from proteolytic processing of domain 1 of its precursor protein, prosaposin. Processing of prosaposin (70 kDa) also generates three other previously reported saposin proteins, B, C, and D, from its second, third, and fourth domains. Similar to saposin C, saposin A stimulates the hydrolysis of 4-methylumbelliferyl beta-glucoside and glucocerebroside by beta-glucosylceramidase and of galactocerebroside by beta-galactosylceramidase, mainly by increasing the maximal velocity of both reactions. Saposin A is as active as saposin C in these reactions. Saposin A has no significant effect on other sphingolipid and 4-methylumbelliferyl glycoside hydrolases tested. Saposin A has two potential glycosylation sites that appear to be glycosylated. After deglycosylation, saposin A had a subunit molecular mass of 10 kDa and was as active as native saposin A. However, reduction and alkylation abolished the activation. A three-dimensional model comparing saposins A and C reveals significant sequence homology between them, especially preservation of conserved acidic and basic residues in their middle regions. Each appears to possess a conformationally rigid hydrophobic pocket stabilized by three internal disulfide bridges, with amphipathic helical regions interrupted by helix breakers.