GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans

Abstract

Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA (cellulase synthase-like protein). In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective, the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

“…Deletion of cslA abolishes pellet formation in liquid-grown cultures and also blocks the formation of reproductive aerial hyphae on solid medium (28,29). GlxA is a newly characterized member of the AA5 family of mono-copper oxidase possessing a Tyr-Cys cross-linked redox cofactor (30). A glxA null mutant blocks development and abolishes pellet formation coinciding with the loss of glycan deposition at hyphal tips, the same phenotype as observed with the cslA deletion (30,33).…”

“…copper-loading and subsequent Tyr-Cys cross-link formation (30)). Based on these studies, a model has been developed that proposes the presence of a large protein complex at the hyphal tip for synthesis and deposition of the glycan required for morphogenesis (36).…”

“…GlxA is a newly characterized member of the AA5 family of mono-copper oxidase possessing a Tyr-Cys cross-linked redox cofactor (30). A glxA null mutant blocks development and abolishes pellet formation coinciding with the loss of glycan deposition at hyphal tips, the same phenotype as observed with the cslA deletion (30,33). This is consistent with a model whereby CslA and GlxA cooperatively function in glycan deposition at the hyphal tip and are jointly responsible for the formation of reproductive aerial structures and, in liquid environments, for the formation of pellets (30).…”

Aggregation of germlings is a major contributing factor towards mycelial heterogeneity of Streptomyces

“…These include among others the recently discovered mat genes11, as well as cslA, glxA and dtpA 121314. The common feature of these genes is that they encode proteins that are involved in the synthesis of extracellular glycans.…”

Recent advances in understanding Streptomyces

“…Continued hyphal elongation is co-ordinated by the DivIVA-based polarisome 50 and (in addition to cell wall growth) involves the extracellular production of cellulose-like glycan catalysed by polarisome-linked ClsA (cellulose synthase-like) associated with the copper oxidase GlxA 52, 53 . MatAB proteins have an uncharacterised role in glycan production 54 .…”