The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.     

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus.

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.     

Purification and characterization of a veratryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus

A veratryl alcohol oxidase (VAO) enzyme was discovered in cultures of Pleurotus ostreatus. The enzyme, which oxidizes veratryl alcohol to veratraldehyde reducing O2 to H2O2, was purified to homogeneity and its main structural and catalytic properties have been determined. The enzyme is a glycoprotein and contains FAD as a prosthetic group. The amino acid composition and carboxy- and amino-terminal sequences were determined. Primary aromatic alcohols with methoxy substituents in position four are good substrates for VAO; cinnamyl alcohol is the substrate which is oxidized faster whereas coniferyl alcohol is oxidized at a slower rate. The enzyme is moderately thermostable (t1/2(55 degrees C) about 1.5 h, apparent melting temperature about 60 degrees C). The enzyme stability in 50% water/organic solvents mixtures has also been studied.     

Secretion of alpha-amylase from Pseudoalteromonas haloplanktis TAB23: two different pathways in different hosts

Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for alpha-amylase secretion in Escherichia coli. The occurrence of two different secretion pathways in different hosts is proposed.     

Structural characterization of heterodimeric laccases from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and heterogeneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants—differing for an insertion of four amino acids—for each isoenzyme. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N-glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known.