Gene cloning and sequencing, and enzyme purification of the malate synthase of Streptomyces arenae

Abstract

Streptomyces arenae is able to grow on acetate or ethanol as the sole carbon source. The metabolic pathway used for gluconeogenesis from C2 compounds in streptomycetes has not yet been characterized. In the course of a sequencing project we identified the gene for malate synthase ( aceB), a key enzyme in the glyoxylate cycle in S. arenae. The gene was cloned and sequenced. The open reading frame of 1632 bp codes for a potential protein of 61.360 kDa. A comparison with the sequences of malate synthase from other organisms shows that the phylogenetic distance to the E. coli aceB gene is no closer than that to genes from plants or fungi. Malate synthase activity was detected in cell extracts from S. arenae. Its dependence on media conditions and on the growth phase was investigated. A purification procedure was established which allows a 188-fold enrichment of the enzyme. The molecular weight of the monomer determined by SDS PAGE confirms the weight calculated from the gene sequence. However, the holoenzyme appears to be dimeric as shown by gel filtration. All other known malate synthases from eubacteria are monomeric, while those of fungi or plants are oligomeric (di-, tri-, tetra- or octameric). The apparent K m value for glyoxylate is significantly higher than that of the malate synthases of all other species published so far. The enzyme is inactive at pH values of 7 and below; the strain cannot grow on ethanol or acetate as the sole carbon source at media pH values of 7 or below. © 1997 Elsevier Science B.V. All rights reserved.