Chemoenzymatic production of 1,5-dimethyl-2-piperidone

A chemoenzymatic process for the preparation of 1,5-dimethyl-2-piperidone (1,5-DMPD) from 2-methylglutaronitrile (MGN) has been demonstrated. MGN was first hydrolyzed to 4-cyanopentanoic acid (4-CPA) ammonium salt using the nitrilase activity of immobilized Acidovorax facilis 72W cells. The hydrolysis reaction produced 4-CPA ammonium salt with greater than 98% regioselectivity at 100% conversion, and at concentrations of 170–210 g 4-CPA/l. Catalyst productivities of at least 1000 g 4-CPA/g dry cell weight (dcw) of immobilized cells were achieved by recycling the immobilized-cell catalyst in consecutive stirred-batch reactions. After recovery of the immobilized cell catalyst for reuse, the 4-CPA ammonium salt in the aqueous product mixture was directly converted to 1,5-DMPD by low-pressure catalytic hydrogenation in the presence of added methylamine.     

Ala-His Mediated Peptide Bond Formation Revisited

The historical origin of the translation machinery remainsunresolved. Although the large 23S ribosomal RNA (rRNA) is almost certainly the catalytic component of the peptidyl transferase center in the modern ribosome, it is likely thatgreatly simplified systems were initially employed in the latestages of the prebiotic world. In particular, it has been suggested that small RNAs carrying amino acids were important forthe genesis of protein synthesis. Consistent with this, adipeptide, Ala-His, was previously claimed to be a prebioticallyfeasible catalyst mediating peptide bond formation in thepresence of aminoacylated tRNA and cognate mRNA template, in theabsence of other ribosomal components (Shimizu, 1996). We hereinreport a detailed study of putative dipeptide formation by Ala-His and RNAs carrying leucine. Based on the results presentedhere, it is unlikely that the dipeptide, Ala-His, catalyzessignificant levels of Leu-Leu dipeptide formation in solution. Aproduct is produced which can be readily mistaken for a dipeptidein the TLC separation systems employed in earlier work. We offerexplanations for the formation of this product as well as anotherunexpected product. The results presented here are consistentwith the notion that the translation machinery was likely basedon catalytic RNA from its very inception.     

Dephosphorylation of synthetic phosphopeptides by protein phosphatase-T, a phosphothreonyl protein phosphatase

The synthetic phosphohexapeptides Arg-Arg-Ala-Thr(35P)-Val-Ala and Arg-Arg-Ala-Ser(32P)-Val-Ala, phosphorylated by the cAMP-dependent protein kinase and differing only in the nature of the phosphorylated residue, have been used as substrates of a partially purified rat liver protein phosphatase-T, distinct from the multifunctional protein phosphatase-1. While the phosphothreonyl hexapeptide is readily dephosphorylated (exhibiting a Km = 15 microM), the phosphoseryl one is almost unaffected. Such a behavior is not shared by protein phosphatase-1, calf intestine alkaline phosphatase, and potato acid phosphatase, all of which are more active on the phosphoseryl hexapeptide. The NH2-terminal basic residues critical for cAMP-dependent phosphorylation are not required in the dephosphorylation reaction, as both Arg can be removed without impairing the efficiency of protein phosphatase-T toward the phosphothreonyl peptide. On the other hand, the replacement of 2 Pro for the Ala and Val flanking Thr(32P), to give a new phosphohexapeptide reproducing the phosphorylated site of protein phosphatase inhibitor-1, prevents the protein phosphatase-T activity. Moreover, IgG heavy chain 32P labeled in tyrosine is not affected by protein phosphatase-T, while it is dephosphorylated by alkaline phosphatase. These results would indicate that protein phosphatase(s)-T represent a distinct class of protein phosphatases specifically involved in the dephosphorylation of phosphothreonyl residues fulfilling definite structural requirements.     

Cyclic GMP and nitroprusside inhibit the activation of human platelets by fluoroaluminate

Sodium nitroprusside, an activator of the soluble guanylate cyclase, inhibits the intracellular Ca2+ mobilization, ATP secretion and aggregation of human platelets evoked by fluoroaluminate. Similar results are obtained with 8-bromo-cyclic GMP (8-Br-cGMP). Both nitroprusside and 8-Br-cGMP inhibit the protein kinase C-dependent phosphorylation of the 47 and 20 kDa proteins induced by fluoroaluminate, but not by the protein kinase C activators phorbol ester and diacylglycerol. Since fluoroaluminate interacts directly with a G protein, the present results suggest that the cGMP interferes with platelet activation at the level of G protein-phospholipase C.