Strictly Polyphosphate-Dependent Glucokinase in a Polyphosphate-Accumulating Bacterium, Microlunatus phosphovorus

Abstract

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P i from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of orthophosphate (P i ) residues linked in the same manner as the two high-energy phosphoanhydride bonds in ATP (4, 5). PolyP is readily formed by dehydration of P i and found in abundance in volcanic condensates and deep-oceanic steam vents (14). Hence, ancient organisms may have utilized polyP instead of ATP in their metabolic reactions (4,9,14).Glucokinases that use ATP as the sole phosphoryl donor to catalyze the phosphorylation of glucose (ATP-GKs) have been present in all contemporary organisms examined (2). Bifunctional glucokinase (polyP/ATP-GK), which utilizes polyP or ATP as the phosphoryl donor to phosphorylate glucose, was found first in Mycobacterium phlei (10) and then in many other bacteria, including Corynebacterium diphtheriae (11), Mycobacterium tuberculosis (12), and Propionibacterium shermanii (13). The polyP/ATP-GK of M. tuberculosis also utilizes GTP, UTP, and CTP as phosphoryl donors (12). PolyP is recognized as one of the earliest biopolymers and is most likely a prominent precursor in prebiotic evolution (4). Thus, it has been hypothesized that glucose phosphorylation was originally mediated by polyP and that when ATP became available in the environment, a transition was made by the GKs to utilize the latter phosphoryl donor (9). However, nobody has discovered the strictly polyP-dependent glucokinase that utilizes polyP as the sole phosphoryl donor (polyP-GK).Microlunatus phosphovorus strain NM-1 is a gram-positive, coccus-shaped, non-spore-forming bacterium (7). Strain NM-1, which was originally isolated with an enhanced biological phosphorus removal process, accumulates large amounts of polyP (a maximum of approximately 48% of its dry weight as phosphate [P i ]) in a glucose medium (7). We discovered the existence of polyP-GK in M. phosphovorus strain NM-1.M. phosphovorus strain NM-1 was grown for 2 days under aerobic conditions at 27°C in the glucose medium (7). The cells harvested at the mid-logarithmic phase of growth were disrupted by using Bead-beater (Biospec), and the polyP-GK was precipitated by adding 70% ammonium sulfate. The polyP-GK was further purified by using a DEAE-cellulose (Whatman), a phenyl-Sepharose (Amersham Biosciences), a PE hydrophobic (Poros), and two anion exchange (HQ [Poros] and miniQ [Amersham Biosciences]) columns. The polyP-GK was purified 253-fold (Fig. 1A), and the recovery of polyP-GK activity was 6.7%. The polyP-GK migrated as a 32-kDa protein on sodium dodecyl sulfate (SDS)-polyacrylamide gels, while...

Crystal Structure of Bacterial Inorganic Polyphosphate/ATP-glucomannokinase

“…2b) were conserved in the primary structures of C-HKI as Thr 536 and Arg 539 (Fig. 5) and in other eucaryotic HKs (13), Gram-negative and -positive bacterial GKs including poly(P)-and poly(P)/ATP-GKs (1,3,6), and even some proteins in the ASKHA superfamily (40,44). The sequence around residues highly conserved in these proteins is called a "phosphate-1" motif (44).…”

“…Note that poly(P)-GK is poly(P)-specific, although poly(P)/ATPtype enzymes use poly(P) and ATP. Although the primary structures of these poly(P)-dependent kinases were determined (1)(2)(3)(4), the lack of a crystal structure has prevented us from clarifying the poly(P)-utilizing mechanism of these unique kinases. Poly(P)-dependent kinases have ATP-specific partners, and a knowledge of the crystal structure of poly(P)-dependent kinase may aid in understanding the structural relationship of poly(P)-dependent kinase to the ubiquitous ATP-specific kinase, i.e.…”

Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

“…The ATP/ polyphosphate glucokinase from Mycobacterium tuberculosis (30) and glucomannokinase from Anthrobacter sp. strain KM (45) as well as the strictly polyphosphate-dependent GlK from Microlunatus phosphovorus (66) are also members of this group.…”

“…Glucokinases that utilize ATP as a phosphoryl donor may, in addition, use other nucleoside triphosphates (37,58) or polyphosphate (reviewed in reference 53) as substrates or both ATP and polyphosphate (30,52). Recently, a strictly polyphosphate-dependent glucokinase (EC 2.7.1.63) has been purified from Microlunatus phosphovorus (66). GlK from E. coli has been cloned, purified, and studied kinetically (43).…”

Enzymes of Inorganic Polyphosphate Metabolism

“…There is a progressive decrease in the efficiency of PolyP utilization by glucokinases, from older to newer organisms (Phillips et al 1999). Some enzymes occurring among glucokinases of different bacteria are able to use for glucose phosphorylation mainly PolyP, like in Microlunatus phosphovorus (Tanaka et al 2003); PolyP and ATP, like in P. shermanii and M. tuberculosis (Phillips et al 1999); or ATP only, like in E. coli. The active center responsible for PolyP utilization was probably eliminated in the course of evolution, and the glucokinases of eukaryotes already cannot use this inorganic substrate.…”