Hiroyuki Sugino

Purification and characterization of monoamine oxidase from Klebsiella aerogenes

Abstract The gene for monoamine oxidase ( maoA ) from Klebsiella aerogenes W70 has been cloned and the enzyme was overproduced in a soluble form. The enzyme was purified approximately 10-fold to homogeneity. The enzyme has a molecular weight of about 79,000, which is identical to the molecular weight deduced from the nucleotide sequence of the gene for monoamine oxidase. The enzyme had maximum activity at pH 6.0 and 50°C when catalyzing the oxidative deamination of tyramine. The enzyme catalyzed the deamination of β-phenylethylamine, dopamine, tryptamine, and octopamine, but not of diamines, polyamines, or amino acids. The enzyme was inhibited by clorgyline, isoniazid, and carbonyl reagents, but not by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The enzyme did not exhibit a typical flavoprotein spectrum, but the enzymatic activity increased linearly with increasing amounts of added copper. The purified enzyme was found to contain 10 g of copper per 79,000 g of the protein. The enzymological properties and the amino acid sequence of the enzyme deduced from the nucleotide sequence of the moaA gene are different from those of known tyramine or monoamine oxidases.

Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei

The 200kb linear plasmid pSLA2-L was suggested to be involved in the production of two macrolide antibiotics, lankamycin (Lm) and lankacidin (Lc), in Streptomyces rochei 7434AN4. Hybridization experiments with the polyketide synthase (PKS) genes for erythromycin and actinorhodin identified two eryAI-homologous regions and an actI-homologous region on pSLA2-L. The nucleotide sequence of a 3.6kb SacI fragment carrying one of the eryAI-homologs revealed that it codes for part of a large protein with four domains for ketoreductase, acyl carrier protein, ketosynthase, and acyltransferase. Gene disruption confirmed that the two eryAI-homologs are parts of a large type-I PKS gene cluster for Lm. A 4.8kb DNA carrying the actI-homologous region contains four open reading frames (ORF1-ORF4) as well as an additional ORF, i.e. ORF5, which might code for a thioesterase. Deletion of the ORF2-ORF4 region showed that it is not involved in the synthesis of Lm or Lc. Thus, it was confirmed that pSLA2-L contains two PKS gene clusters for Lm and an unknown type-II polyketide.

Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from Klebsiella aerogenes

SummaryWe cloned the structural gene for monoamine oxidase (maoA) from Klebsiella aerogenes into a pKI212 vector in an maoA mutant strain of K. aerogenes. Deletion analysis and complementation tests of the recombinant plasmid showed that the maoA gene was located entirely within a 4.1-kb segment. In an maoA mutant strain harbouring the cloned maoA gene, synthesis of monoamine oxidase was induced by addition of tyramine and related compounds. Transfer of a plasmid containing the maoA gene into a monoamine oxidase-producing strain of K. aerogenes W70 resulted in about a 30- to 40-fold increase in total production of the enzyme. When cells of K. aerogenes carrying the plasmid containing the maoA gene were grown with tyramine, more than 85% of the monoamine oxidase was produced in soluble form, whereas the parent strain W70 produced most monoamine oxidase as the membrane-bound form.

A Klebsiella aerogenes moaEF operon is controlled by the positive MoaR regulator of the monoamine regulon.

A 30-kDa protein accumulated upon induction by a high concentration of tyramine or dopamine in cells of Klebsiella aerogenes (Ka). These cells carried a plasmid (pAS123) that included the arylsulfatase operon (atsBA). Deletion analysis showed that the region essential for induction of the 30-kDa protein was located within a 2.0-kb cloned segment downstream of the atsBA operon. The nucleotide (nt) sequence of the 2.0-kb fragment revealed two open reading frames (ORFs), moaE and moaF. Transcription from a putative promoter of moaE was induced by the addition of tyramine, and the moaF gene was co-transcribed from this monoamine-inducible Ka promoter. The deduced Ka MoaE protein was homologous to insect-type alcohol dehydrogenase. The sequence of the 18 amino acids from the N-terminus of the purified 30-kDa protein agreed with that deduced from the nt sequence of moaF. Using a Ka strain with a mutant moaR gene, we found that MoaR, that acts as the positive regulator of the monoamine regulon, also acts as the positive regulator of the moaEF operon.

Gene Targeting in a Klebsiella sp. by Fusaric Acid Selection and the Use of Temperature Sensitive Replicon

Two previously described methods for gene targeting (replacement) in Escherichia coli were applied to the disruption of the maoCA operon in Klebsiella aerogenes. These techniques involve plasmid-chromosomal integration, resolution of the integrated intermediate and segregation (i) screened using fusaric acid for the counter selection of plasmid replicons carrying the gene for tetracycline resistance, or (ii) by using a temperature sensitive replicon. Both methods were found to be effective to create the desired chromosomal mutants in a Klebsiella strain after some modifications of the original experimental protocols, and may serve as tools for gene targeting studies in Klebsiella and related species.