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Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-D-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-DL-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.

Research has been conducted on the microbial phytosterol degradation. Degradation of these sreroid intermediates initiated by Delta (super)1 -dehydrogenation of the steroid ring structure has been investigated.

The kstD gene which encodes a 3-ketosteroid delta(super.1)-dehydrogenase isoenzyme of Rodococcus erythropolis strain SQ1 and its targeting disruption are discussed. R. erythropolis was shown to have two activity bands, one of which is not seen in the kstD mutant strain. Inactivation of kstD is not sufficient for inactivation of the delta(super.1) dehydrogenase activity.

A gene library of Amycolatopsis methanolica total DNA in Escherichia coli with a pMEA300-derived shuttle vector was devised and the characteristics of the pdt gene were studied. Nucleotide sequencing showed that the pdt gene has a single large open reading frame that codes for 304 amino acids. Its deduced amino acid sequence was very similar to the N-terminal amino acid sequence of the A. methanolica PDT protein.

The pMEA300 plasmid of the Amycolatopsis methanolica responsible for conjugational transfer was identified and its nucleotide sequence and function were studied. The TraG, TraH, TraI, and TraJ proteins were needed to effectively transport pMEA300. Two putative proteins, TraA and TraB, were found implicated in pock development. The pMEA300-encoded KorA protein binds particularly with the tra-korA intragenic region.

An analysis of the plasmid pMEA300 found in Amycolatopsis methanolica NCIB 11946 shows that it is 13.3 kb long and exists both in the free state and within a bigger replicon. A nucleotide sequencing of the NspH1 segment of pMEA300 showed that it contains two amino-acid sequences, the first of which is similar to the Xis protein of plasmids such as pSAM2, and the second similar to the C terminus of proteins of the Int site-specific recombinases. The attP site is 44bp in length.

Investigation of the enzymes associated with glucose and methanol metabolism in actinomycete Amycolatopsis methanolica reveals that positive effectors of ATP-dependent phosphofructokinase (PFK) exert no influence on the A. methanolica PPi-dependent PFK, but N-terminal amino acid sequence of PPi-PFK exhibits homology with ATP-PFK enzymes from other bacteria. Purification and analysis of methanolica pyruvate kinase reveals it to be an allosteric enzyme. A study with mutants and protein purifications reveals the involvement of pentose phosphate pathway transaldolase enzyme in growth on glucose and an additional Cl-inducible, ribulose monophosphate cycle transaldolase isoenzyme in growth on methanol.

The purification and characterization of a Type II 3-dehydroquinate dehydratase from the actinomycete Amycolatopsis methanolica were reported. It was shown that the organism grows rapidly on quinate but not on shikimate as sole carbon and energy source. Enzyme studies and analysis of mutants showed that the A. methanolica 3-dehydroquinate dehydratase is involvedin both the catabolism of quinate and the biosynthesis of aromatic amino acids.Its characterization indicated that it is a heat-stable and oligomeric protein with a subunit M(sub r) of 12 x 10(super 13).