Functional consequences of posttranslational isomerization of Ser46 in a calcium channel toxin.

The venom of the funnel-web spider Agelenopsis aperta contains several peptides that paralyze prey by blocking voltage-sensitive calcium channels. Two peptides, omega-Aga-IVB (IVB) and omega-Aga-IVC (IVC), have identical amino acid sequences, yet have opposite absolute configurations at serine 46. These toxins had similar selectivities for blocking voltage-sensitive calcium channel subtypes but different potencies for blocking P-type voltage-sensitive calcium channels in rat cerebellar Purkinje cells as well as calcium-45 influx into rat brain synaptosomes. An enzyme purified from venom converts IVC to IVB by isomerizing serine 46, which is present in the carboxyl-terminal tail, from the L to the D configuration. Unlike the carboxyl terminus of IVC, that of IVB was resistant to the major venom protease. These results show enzymatic activities in A. aperta venom being used in an unprecedented strategy for coproduction of necessary neurotoxins that possess enhanced stability and potency.

Biosynthesis of D-amino acid-containing peptides: exploring the role of peptide isomerases.

The discovery of D-amino acid residues in a growing number of gene-encoded peptides suggests that such biochemical modifications are more common than initially thought. In fact, the extent to which D-amino acids are incorporated into peptides by multicellular organisms probably has not been fully realized, since routine Edman sequencing does not provide the absolute stereochemistry of amino acid residues. Unless both the D and L isomers of a particular peptide sequence are isolated, D-amino acid-containing peptides are often identified only after synthesis of naturally-occurring peptide fails to yield the desired activity. To date, D-amino acid residues (e.g., alanine, methionine, leucine, isoleucine, phenyl alanine, asparagine, tryptophan and serine) have been identified in peptides from a variety of species, including frogs, snails, clams, lobsters and spiders. While most have a single D-amino acid residue located near their N-termini, an exception is found with omega-Aga IVB. The examples highlighted in this chapter are the result of a unique strategy of multicellular organisms to circumvent stereochemical limitations imposed by the genetic code in an effort to increase molecular diversity. The presence of D-amino acids permits the generation of novel tertiary structure that could not be accessed from L-amino acids alone. Moreover, advantages of increased potency and protease stability are often observed. Our understanding of the biosynthesis of these D-amino acid-containing peptides is still in its infancy. Nevertheless, the discovery of a novel peptide isomerase from the venom of the Agelenopsis aperta spider provides some important clues to explain the incorporation of single D-amino acid residues within a peptide chain. Given its high homology with other serine proteases, the isomerase may represent an opportune mutation in response to evolutionary pressures. Yet, is the isomerase a unique exception or simply the first in a class of enzymes of varying substrate specificity capable of synthesizing D-amino acid-containing peptides? To be sure, much more remains to be explored about the precise timing and mechanism of the isomerization process, in addition to obtaining further structural data on the enzyme itself. Therein lies the continuation of this fascinating story in enzyme biochemistry.

Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains.

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3.

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.