bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.448987; this version posted June 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The ADP-glucose pyrophosphorylase from Melainabacteria: a comparative 2 study between photosynthetic and non-photosynthetic bacterial sources 3 4 María V. Ferrettia, Rania A. Hussienb,c, Miguel A. Ballicorab, 5 Alberto A. Iglesiasa, Carlos M. Figueroaa, Matías D. Asencion Dieza§ 6 7 aInstituto de Agrobiotecnología del Litoral, Universidad Nacional del Litoral, Consejo 8 Nacional de Investigaciones Científicas y Técnicas, Facultad de Bioquímica y Ciencias 9 Biológicas, Santa Fe, Argentina 10 bDepartment of Chemistry and Biochemistry, Loyola University Chicago, Chicago, Illinois, USA 11 cDepartment of Chemistry, Al Baha University, P.O. Box 1988. Al Baha, Saudi Arabia Imam 12 Mohammed Ibn Saud Islamic University, Riyadh, Saudi Arabia 13 14 15 §Corresponding author: 16 Dr. Matías D. Asencion Diez [email protected] 17 Instituto de Agrobiotecnología del Litoral (UNL-CONICET). CCT-Santa Fe, Colectora 18 Ruta Nac 168 km 0. Santa Fe (3000), Argentina. 19 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.448987; this version posted June 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Abstract 21 Until recently, all members of the cyanobacterial phylum were considered capable of 22 performing oxygenic photosynthesis. This view has been questioned after the discovery of 23 a group of presumed non-photosynthetic cyanobacteria named Melainabacteria. Using 24 metagenomic data, we identified sequences encoding putative ADP-glucose 25 pyrophosphorylase (EC 2.7.7.27, ADP-GlcPPase) from free-living and intestinal 26 Melainabacteria. These genes were de novo synthesized and overexpressed in Escherichia 27 coli. The purified recombinant proteins from the free-living and the intestinal 28 Melainabacteria showed ADP-GlcPPase activity, with Vmax values of 2.3 and 7.1 U/mg, 29 respectively. Both enzymes had similar affinities towards ATP (S0.5 ~0.3 mM) although the 30 one from the intestinal source displayed a 6-fold higher affinity for glucose-1P. Both 31 recombinant ADP-GlcPPases were sensitive to allosteric activation by glucose-6P (A0.5 32 ~0.3 mM), and to inhibition by Pi and ADP (I0.5 between 0.2 to 3 mM). Interestingly, the 33 enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the 34 principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. To the best of 35 our knowledge, this is the first biochemical characterization of an active enzyme from 36 Melainabacteria, offering further data to discussions regarding their phylogenetic position. 37 This work contributes to a better understanding regarding the evolution of allosteric 38 mechanisms in ADP-GlcPPases, an essential enzyme for the synthesis of glycogen in 39 prokaryotes and starch in plants. 40 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.448987; this version posted June 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 Introduction 42 Most living organisms produce α-1,4-glucans as a strategy to store carbon and 43 energy, which can be mobilized under conditions of nutrient deficiency. Bacteria and 44 heterotrophic eukaryotes accumulate glycogen, whereas starch is the reserve carbohydrate 45 in green algae and higher plants [1]. The build-up of glycogen and starch in bacteria and 46 plants, respectively, involves a similar pathway where ADP-glucose (ADP-Glc) is the 47 glycosyl donor for polysaccharide elongation. In such metabolic route, the sugar nucleotide 48 synthesis is the rate-limiting step, catalyzed by ADP-Glc pyrophosphorylase (ADP- 49 GlcPPase, EC 2.7.7.27). Indeed, ADP-GlcPPase is allosterically regulated by metabolites 50 from the central carbon utilization pathway in the respective organism [1–4]. In this 51 context, the enzyme from Escherichia coli is mainly activated by fructose-1,6- 52 bisphosphate, a key intermediate in the Embden-Meyerhof route. At the same time, 53 fructose-6-phosphate (Fru-6P) and pyruvate are the principal activators of the enzyme from 54 Agrobacterium tumefaciens, where the Entner-Doudoroff is the main glycolytic pathway 55 [5–9]. Similarly, ADP-GlcPPases from organisms performing oxygenic photosynthesis 56 (cyanobacteria, green algae, and higher plants) are primarily activated by 3- 57 phosphoglycerate (3-PGA) and inhibited by inorganic orthophosphate (Pi) [1,10–12]. 58 Cyanobacteria are a highly diverse group of Gram-negative prokaryotes that 59 colonized a wide range of environments, from desert crusts to fresh and marine waters and 60 from the tropics to the poles [13]. These microorganisms modified the Earth’s atmosphere 61 through oxygenic photosynthesis, which enabled the evolution of life into more complex 62 forms [14]. Photosynthetic cyanobacteria have been studied for decades, and their diversity 63 is described in terms of both morphology and genetics [15–17]. With the development of 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.448987; this version posted June 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 64 metagenomics, an unexpected variety of organisms was unveiled in many ecosystems, 65 including non-photosynthetic bacteria closely related to the clade Cyanobacteria [18–21]. 66 One group of these microorganisms was named Melainabacteria [19] because several 67 representatives of this cluster were found in aphotic environments. Firstly, they were 68 considered as a sister phylum of Cyanobacteria [19]; later, data from genomic sequences 69 analysis suggested that Melainabacteria belongs to the superphylum of Cyanobacteria [20], 70 although these suggestions have not been validated so far. Based on this evidence, a new 71 classification has been proposed for the phylum Cyanobacteria. This arrangement includes 72 the class-level lineages Oxyphotobacteria (cyanobacteria performing oxygenic 73 photosynthesis) and Melainabacteria, as well as a third class called ML635J-21 [20], 74 recently named Sericytochromatia [22]. After diverging from Melainabacteria, the 75 Oxyphotobacteria developed oxygenic photosynthesis around 2.4–2.35 billion years ago, as 76 estimated from the molecular clock and geological data [22–24]. 77 Melainabacteria, described for the first time only a few years ago, is a group of 78 poorly characterized anaerobic bacteria. To date, only a handful of Melainabacteria 79 genomes have been sequenced [22], and thus, the metabolism, biological functions, and 80 ecological roles of these organisms are not fully known. Representatives of 81 Melainabacteria have been found in photic and aphotic environments such as (i) sub- 82 surface groundwater [19], (ii) lake water and algal biofilms [22,25], (iii) marine and 83 lacustrine sediment [26], and (iv) animal and human faeces [20] and guts [26]. All 84 sequenced genomes of Melainabacteria confirm they lack the entire photosynthetic 85 apparatus, which may support the hypothesis that acquisition of photosystems in 86 Oxyphotobacteria occurred after divergence from the non-photosynthetic Melainabacteria 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.448987; this version posted June 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 87 [20,22]. Consequently, the characterization of enzymes from main metabolic segments 88 (such as the synthesis of the energy/carbon storage molecule glycogen) is critical to sum 89 biochemical criteria to help the scientific community to further classify this group of 90 bacteria. 91 By studying the biochemical properties of cyanobacterial ADP-GlcPPases, an 92 evolutionary thread could be established between bacterial glycogen and starch synthesis 93 metabolism [1,2,27,28]. Hence, the particular regulatory properties of ADP-GlcPPases 94 prompted us to explore the features of this enzyme in Melainabacteria. In this framework, 95 we de novo synthesized the genes encoding ADP-GlcPPases from intestinal (inMelGlgC) 96 and free-living (flMelGlgC) Melainabacteria. The recombinant proteins were produced, 97 purified, and kinetically characterized. For the sake of comparison, we also made the 98 homologous enzyme from photosynthetic Anabaena PCC 7120 (AnaGlgC). Our results 99 indicate that Melainabacteria ADP-GlcPPases have distinctive kinetic and regulatory 100 properties, which might fit a heterotrophic metabolism commonly found in diverse bacterial 101 organisms. 102 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.18.448987; this version posted June 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 103 Material and Methods 104 Chemicals, bacterial strains and plasmids 105 Chemicals used for enzymatic assays were from Sigma-Aldrich (St. Louis, MO, 106 USA). All the other reagents were of the highest quality available. Escherichia coli Top 10 107 (Invitrogen) were used for plasmid maintenance. The glgC genes from Anabaena PCC 108 7120, intestinal and free-living Melainabacteria were expressed in E.