Identification of protein structural elements responsible for the diversity of sequence preferences among Mini-III RNases.

Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. In some cases, selectivity is also determined by the structural features of the substrate. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro.

Sequence-specific cleavage of dsRNA by Mini-III RNase.

Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner.

Heterogeneity of quaternary structure of glucosamine-6-phosphate deaminase from Giardia lamblia.

The oligoHis-tagged versions of glucosamine-6-phosphate deaminase from Giardia lamblia (GlmNagB-HisN, GlmNagB-HisC) were constructed and purified to hear homogeneity, and their kinetic and structural properties were compared to those of the wild-type enzyme (GlmNagB). Introduction of the oligoHis tag at the GlmNagB C-terminus resulted in almost complete loss of the catalytic activity, while the catalytic properties of GlmNagB-HisN and GlmNagB were very similar. The recombinant and wild-type enzyme exhibits heterogeneity of the quaternary structure and in solution exists in three interconvertible forms, namely, monomeric, homodimeric, and homotetrameric.

Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification.

Rationally designed muteins of Candida albicans glucosamine-6-phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versions containing internal oligoHis fragments were constructed: (i) by substituting residues 343-348 of the interdomain undecapeptide linker with hexaHis, (ii) by replacing solvent-exposed residues 655-660 of the isomerase domain with hexaHis, and (iii) by replacing amino acids at positions 568 and 569 with His residues to generate the three-dimensional hexaHis microdomain in the enzyme quaternary structure. The resulting constructs were effectively purified to near homogeneity by rapid, one-step immobilized metal-ion affinity chromatography and demonstrated activity and catalytic properties comparable with that of the wild-type enzyme.

N gamma-alkyl derivatives of L-glutamine as inhibitors of glutamine-utilizing enzymes.

A general, facile method to synthesize the N gamma-alkyl and N gamma,N gamma-dialkyl derivatives of L-glutamine 1a-d from L-glutamic acid as a starting substrate is presented. The obtained compounds are shown to inhibit three different glutamine-utilizing enzymes, namely: glutaminase, gamma-glutamyl transpeptidase, and glucosamine-6-phosphate synthase, with inhibitory constants within the millimolar range.