Molecular fingerprints for a novel enzyme family in actinobacteria with glucosamine kinase activity

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production. IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature’s main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.We acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation facilities and thank their staff for help with data collection. Part of these experiments were performed at beamline BL13-XALOC of ALBA Synchrotron (Cerdanyola del Vallès, Spain), with the collaboration of ALBA staff and CALIPSOplus (grant 730872) funding. The support of the X-ray Crystallography Scientific Platform of i3S (Porto, Portugal) is also acknowledged. We thank Pedro Lamosa from CERMAX, ITQB-NOVA (Oeiras, Portugal), for acquiring and interpreting the NMR data. This work was supported by the Structured Program on Bioengineered Therapies for Infectious Diseases and Tissue Regeneration (Norte-01-0145-FEDER-‏000‏012), funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through Fundo Europeu de Desenvolvimento Regional (FEDER) and by FEDER through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274) and also by grants UID/NEU/04539/2019 and POCI-01-0145-FEDER-029221. D.N.-C. acknowledges the European Regional Development Fund (CENTRO-01-0145-FEDER-000012-ealthyAging2020) for a research fellowship and FCT for Ph‏D f‏ellowship SFRH/BD/117777/2016