Bacteria belonging to the Streptomyces genus are characterized by a complex life cycle and the production of many bioactive secondary metabolites. Trace metals play an important role in streptomycete metabolism and development, however, their mechanism of action is not fully understood. In this review, we summarize the present knowledge on metallosensing regulators and trace metal action, as well as discuss the possible application in natural product discovery.
It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.
Penicillins and cephalosporins are β-lactam antibiotics widely used to treat bacterial infectious diseases. They mainly target the cell wall biosynthesis pathway to inhibit bacterial growth. The targets, known as penicillin-binding proteins, are enzymes involved in the polymerization of glycan chains, cross-linking them during bacterial cell wall formation. However, the dispensation of these antibiotics has been concomitant with increasing incidence of resistance to them. Reportedly, this is due to the evolvement of two resistance mechanisms in the bacterial pathogens. One is the production of β -lactamases that cleave the β -lactam rings of penicillin and cephalosporin antibiotics, rendering them ineffective against the pathogens. Another is the modification of the targets, resulting in their inability to bind β -lactam antibiotics. Nevertheless, β -lactam antibiotics remain clinically relevant due to their high target specificity in bacteria and low toxicity to humans. Thus, to overcome the continuing emergence of resistance in pathogens, more efficacious β -lactams have to be developed and cephalosporins are often preferred over penicillins due to two alkyl sites in the cephalosporin core structure amenable for modification. Transformed β -lactams are expected to have improved antimicrobial spectra and pharmacokinetics. This is illustrated by the development of two cephalosporins, namely ceftobiprole and ceftaroline, which have shown good antimicrobial activities and are currently undergoing clinical trials. This review will discuss computer-aided studies of three enzymes closely related to cephalosporins: (1) its synthesizing enzyme, deacetoxycephalosporin C synthase, (2) its targets, the penicillin-binding proteins, and (3) its degrading enzyme, the β -lactamases, and their implications in the development of new cephalosporins.
The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.
It is well known that the co-chaperone p23 regulates Hsp90 chaperone activity in protein folding. In Plasmodium falciparum, a putative p23 (Pfp23) has been identified through genome analysis, but its authenticity has remained unconfirmed since co-immunoprecipitation experiments failed to show its interaction with P. falciparum Hsp90 (PfHsp90). Thus, recombinant Pfp23 and PfHsp90 proteins purified from expressed clones were used in this study. It was clear that Pfp23 exhibited chaperone activity by virtue of its ability to suppress citrate synthase aggregation at 45 degrees C. Pfp23 was also shown to interact with PfHsp90 and to suppress its ATPase activity. Analyses of modeled Pfp23-PfHsp90 protein complex and site-directed mutagenesis further revealed strategically placed amino acid residues, K91, H93, W94 and K96, in Pfp23 to be crucial for binding PfHsp90. Collectively, this study has provided experimental evidence for the inherent chaperone function of Pfp23 and its interaction with PfHsp90, a sequel widely required for client protein activation.
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