Regulatory Properties of the ADP-Glucose Pyrophosphorylase from the Clostridial Firmicutes Member Ruminococcus albus

Abstract

ADP-glucose pyrophosphorylase from Firmicutes is encoded by two genes (glgC and glgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups of Firmicutes, Bacillales and Lactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase from Clostridiales, the third group in Firmicutes, has been characterized. For this reason, we cloned the glgC and glgD genes from Ruminococcus albus Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of the Bacillales enzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases from Firmicutes were unregulated. Our results, together with data from Lactobacillales, indicate that heterotetrameric Firmicutes enzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation.IMPORTANCE The enzymes involved in glycogen synthesis from Firmicutes have been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase from Ruminococcus albus Our results showed that this protein that belongs to different groups from Firmicutes (Bacillales, Lactobacillales, and Clostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in the Firmicutes group, whereby we propose that these heterotetrameric enzymes, with the exception of Bacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family in Firmicutes.

Keywords: ADP-glucose pyrophosphorylase; GlgC; GlgD; Ruminococcus albus; allosterism; fructose-1,6-bisphosphate; glycogen; glycogen metabolism; phosphoenolpyruvate; pyruvate.

FIG 1

Expression of genes coding for R. albus ADP-Glc PPase in E. coli AC70R1-504 cells. (A) Specific ADP-Glc PPase activity in crude extracts of cells transformed with the following plasmids: none (control), pMAB5/RalglgC (GlgC), pMAB6/RalglgD (GlgD), and pMAB5/RalglgC plus pMAB6/RalglgD (GlgC/GlgD). The basal activity was 0.03 ± 0.01 mU/mg. (B and C) Immunodetection of the R. albus ADP-Glc PPase subunits in soluble crude extracts, stained using as primary antibodies those raised against the G. stearothermophilus GlgC and GlgD subunits.

FIG 2

Functionality of the genes coding for the R. albus ADP-Glc PPase. (A) Iodine staining of cell pellets from E. coli AC70R1-504: nontransformed (control) or transformed to produce GlgC, GlgD, or GlgC/GlgD proteins from G. stearothermophilus (Gst), R. albus (Ral), or S. mutans (Smu). (B) Glycogen accumulation in extracts from cells transformed with glgC (black bars), glgD (white bars), or glgC/glgD (gray bars) genes. Glycogen accumulation in control cells was 13.65 ± 0.01 μg of glycogen/OD₆₀₀. For S. mutans ADP-Glc PPase production, cells were transformed with pMAB6/SmuglgC and pMAB5/SmuglgD vectors as described previously (8).

FIG 3

Sensitivity to allosteric regulators of active structures of R. albus ADP-Glc PPase. Responses of GlgC/GlgD (open symbols) and GlgC (filled symbols) to different concentrations of Pyr (A), PEP (B), and Fru-1,6-P₂ (C) are depicted. Values of relative activity were calculated as the ratio of activity determined at each specific condition related to the activity of the respective enzyme form assayed in the absence of effector: 0.87 ± 0.01 U/mg for GlgC and 2.1 ± 0.1 U/mg for GlgC/GlgD.

FIG 4

Inhibitory effect of NAD(P)H in R. albus ADP-Glc PPase GlgC (A) and GlgC/GlgD (B) conformations. Inhibition curves were determined at subsaturating substrate concentrations. The value 1 equals 0.11 U/mg and 0.38 U/mg for GlgC or GlgC/GlgD, respectively. Filled symbols correspond to NADH (■) and NADPH (●), while empty symbols correspond to NAD⁺ (□) and NADP⁺ (○).

FIG 5

Relative catalytic efficiency of R. albus GlgC and GlgC/GlgD in the absence (black bars) or in the presence of 50 mM Pyr (right diagonal bars), 12 mM PEP (gray bars), 12 mM Fru-1,6-P₂ (white bars), and 5 mM NADH (left diagonal bars) for GlgC/GlgD. Relative catalytic efficiency was established as the ratio between the catalytic efficiency (k_cat/S_0.5) obtained under each condition and that, respectively, determined in the absence of effector for GlgC (5.80 s⁻¹ mM⁻¹ and 1.74 s⁻¹ mM⁻¹ for Glc-1P and ATP, respectively) and GlgC/GlgD (22.4 s⁻¹ mM⁻¹ and 8.77 s⁻¹ mM⁻¹ for Glc-1P and ATP, respectively).

FIG 6

Phylogenetic tree of GlgC and GlgD from Firmicutes. Sequences of the GlgC and GlgD polypeptides were collected and the tree was built as described in Materials and Methods. Sequences are numbered with codes indexed in Table S2 in the supplemental material. Different colors represent distinct taxonomy as noted; black indicates sequences taken as reference, and different clusters of Clostridiales are indicated by different shades.