Jan W. Vrijbloed

Targeted disruption of the kstD gene encoding a 3-ketosteroid Delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1

ABSTRACT Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Δ1-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment ofRhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Δ1-dehydrogenase genes. Heterologous expression ofkstD yielded 3-ketosteroid Δ1-dehydrogenase (KSTD) activity under the control of the lac promoter inEscherichia coli. Targeted disruption of thekstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9α-hydroxy-4-androstene-3,17-dione was not abolished by thekstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ1-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation ofkstD is not sufficient for inactivation of the Δ1-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed thatR. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

Purification and characterization of a dual function 3-dehydroquinate dehydratase from Amycolatopsis methanolica

Studies on hydroaromatic metabolism in the actinomycete Amycolatopsis methanolica revealed that the organism grows rapidly on quinate (but not on shikimate) as sole carbon- and energy source. Quinate is initially converted into the shikimate pathway intermediate 3-dehydroquinate by an inducible NAD(+)-dependent quinate/shikimate dehydrogenase. 3-Dehydroquinate dehydratase subsequently converts 3-dehydroquinate into 3-dehydroshikimate, which is used partly for the biosynthesis of aromatic amino acids, and is partly catabolized via protocatechuate and the beta-ketoadipate pathway. Enzyme studies and analysis of mutants clearly showed that the single 3-dehydroquinate dehydratase present in A. methanolica has a dual function, the first example of a 3-dehydroquinate dehydratase enzyme involved in both the catabolism of quinate and the biosynthesis of aromatic amino acids. This enzyme was purified over 1700-fold to homogeneity. Its further characterization indicated that it is a Type II 3-dehydroquinate dehydratase, a thermostable enzyme with a large oligomeric structure (native M(r) 135 x 10(3)) and a subunit M(r) of 12 x 10(3). Characterization of aromatic amino acid auxotrophic mutants of A. methanolica suggested that genes encoding 3-dehydroquinate synthase and 3-dehydroquinate dehydratase are genetically linked but their transcription results in the synthesis of two separate proteins.

A family of macrocyclic antibiotics with a mixed peptide–peptoid ß-hairpin backbone conformation

Macrocyclic peptidomimetics having a mixed peptide-peptoid backbone have been synthesized and shown to possess antibiotic activity against gram-positive and -negative bacteria with a low hemolytic activity against human erythrocytes; one is shown to adopt a regular beta-hairpin conformation by NMR in aqueous solution.

Molecular, Biochemical, and Functional Characterization of a Nudix Hydrolase Protein That Stimulates the Activity of a Nicotinoprotein Alcohol Dehydrogenase

The cytoplasmic coenzyme NAD+-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicusduring growth on C1-C4 primary alcohols is a decameric protein with 1 Zn2+-ion and 1–2 Mg2+-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg2+-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD+-dependent activity of MDH with C1–C4 primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg2+-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. Theact gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD+ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent Km values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 μM, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent Ki values of 3, 160 and 180 μM, respectively. In addition, tryptophan and E4P (apparent Ki values of 11 and 530 μM, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.

Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18 373 Da) and 416 amino acids (45 260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12 917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid‐encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kil—kor system in autonomous replication of pMEA300.

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

SummaryFurther steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14.