Glucosamine-6P and glucosamine-1P, respectively an activator and a substrate of rhodococcal ADP-glucose pyrophosphorylases, show a hint to ascertain (actino)bacterial glucosamine metabolism

Abstract

17Rhodococcus spp. are important microorganisms for biotechnological purposes, such as 18 bioremediation and biofuel production. The latter, founded on the oleaginous characteristic 19 (high lipid accumulation) exhibited by many Rhodococcus species when grown in certain 20 carbon sources under low nitrogen availability. These bacteria accumulate glycogen during 21 exponential growth, and the glucan plays a role as an intermediary metabolite for temporary 22 carbon storage related to lipid metabolism. The kinetic and regulatory properties of the ADP-23 glucose pyrophosphorylase (ADP-GlcPPase) from Rhodococcus jostii supports this 24 hypothesis. The enzyme was found able to use glucosamine-1P as an alternative substrate. 25 Curiously, the activity with glucosamine-1P was sensitive to glucose-6P, the main activator of 26 actinobacterial ADP-GlcPPases. Herein, we report the study of glucosamine-1P related to the 27 activity and regulation of ADP-GlcPPases from R. jostii and R. fascians, with the finding that 28 glucosamine-6P is also a significant activator. Glucosamine-6P, belonging to a node between 29 carbon and nitrogen metabolism, was identified as a main regulator in Actinobacteria. Thus, 30 its effect on rhodococcal ADP-GlcPPases reinforces the function proposed for glycogen as 31 temporary carbon storage. Results indicate that the activity of the studied enzymes using 32 glucosamine-1P as a substrate responds to the activation by several metabolites that improve 33 their catalytic performance, which strongly suggest metabolic feasibility. Then, studying the 34 allosteric regulation exerted on an alternative activity would open two scenarios for 35 consideration: (i) the existence of new molecules/metabolites yet undescribed, and (ii) 36 evolutionary mechanisms underlying enzyme promiscuity that give rise new metabolic features 37 in bacteria.38 39 58 Glycogen metabolism in bacteria depends on the availability of ADP-glucose (ADP-Glc), the 59 specific glucosyl donor in its elongation. The sugar nucleotide is produced by ADP-Glc 60 pyrophosphorylase (EC 2.7.7.27, ADP-GlcPPase), the rate-limiting enzyme in the glucan 61 biosynthesis [7,8]. Except for the case of Bacillus spp., ADP-GlcPPases from all sources 62 characterized so far are allosteric enzymes regulated by key metabolites from the central carbon 63 pathway(s) in the respective organism [7,8]. Depending on the metabolite behaving as a 64 4regulator, ADP-GlcPPases were separated into nine groups [7], although such a classification 65 is incomplete since it lacked data from Gram-positive organisms. We recently fill that gap by 66 characterizing the enzyme from Actinobacteria (Gram-positive with high GC content DNA) 67 and Firmicutes, finding distinctive allosteric effectors [6,[9][10][11][12], which could add groups to that 68 classification. Particularly, we established that glucose-6P (Glc-6P) is the primary activator in 69 the ADP-GlcPPase from Actinobacteria and NADPH is a crucial inhibitor, thus connecting 70 fatty acids synthesis with ca...