A β‐lysine adenylating enzyme and a β‐lysine binding protein involved in poly β‐lysine chain assembly in nourseothricin synthesis in Streptomyces noursei

Abstract

Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly b-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M r 56 000 that speci®cally activates b-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its¯ank-ing DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a b-lysine binding enzyme (NpsB) of about M r 64 100 which can be loaded by NpsA with b-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating b-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly b-lysine chain.

Deciphering Tuberactinomycin Biosynthesis: Isolation, Sequencing, and Annotation of the Viomycin Biosynthetic Gene Cluster

“…We predict that VioO will activate and tether ␤-lysine to its C-terminal PCP domain. Consistent with this proposal, the A domain of VioO has the specificity code for ␤-lysine (DTEDVGTMVK [23]), and preliminary results with partially purified VioO suggest that it activates ␤-lysine (Y. Chan and M. Thomas, unpublished data).…”

Inhibition of Epsilon-Poly- l -Lysine Biosynthesis in Streptomycetaceae Bacteria by Short-Chain Polyols

“…Currently, only the biosynthesis of the poly-␤-lysine chain of streptothricin fits under this classification, although this peptide is not free but is associated with a D-gulosamine core. The gene encoding the ␤-lysine-adenylating enzyme involved in poly-␤-lysine chain assembly is well characterized (4,6). The deduced amino acid sequence exhibits homology with other NRPS family members.…”

A Novel Enzyme Conferring Streptothricin Resistance Alters the Toxicity of Streptothricin D from Broad-spectrum to Bacteria-specific

“…Strains-Based on the highly conserved amino acid sequence of the NATs of S. lavendulae (16), S. rochei (17), and S. noursei (18,19), we designed primers 5Ј-…”

“…1). In fact, in ST producers such as S. lavendulae (16), Streptomyces rochei (17), and Streptomyces noursei (18,19), the ST-resistance genes encoding N-acetyltransferase (NAT) have been identified and their role in selfresistance against their own STs has been investigated. Based on this resistance mechanism and the fact that streptothricin D (ST-D) is a more effective antibiotic than streptothricin F (ST-F), the moiety of ␤-lysine(s) has been shown to play a crucial role in antibiotic activity.…”