Structure and polymerization dynamics of bacterial actin MreB3 and MreB5 involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. It polymerizes into an antiparallel double-stranded filament that generally functions for cell shape determinations by maintaining the cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are responsible for its swimming motility. SpeMreB5 is likely responsible for generating the driving force for the swimming motility. However, molecular profiles involved in the swimming motility are poorly understood. Additionally, SpeMreB3 has distinct sequence features from the other SpeMreBs. Here, we have revealed the structures and polymerization dynamics of SpeMreB3 and SpeMreB5. Both SpeMreBs formed antiparallel double-stranded filaments with different characters; SpeMreB3 formed short filaments with slow polymerization, and SpeMreB5 filaments further assembled into bundle structures such as raft and paracrystal. SpeMreB5 filaments hydrolyzed ATP at a constant rate and were depolymerized immediately after ATP depletion. The Pi release rate of SpeMreB3 was much slower than that of SpeMreB5. Our crystal structure of SpeMreB3 and Pi release measurements of SpeMreB3 and SpeMreB5 mutant variants explain that the cause of the slow Pi release is the lack of the amino acid motif "E ... T - X - [DE]", found in almost all MreBs, which probably takes roles to adjust the position and eliminate a proton of the putative nucleophilic water for γ-Pi of AMPPNP. These results show that SpeMreB3 has unique polymerization dynamics without bundle formations, whereas SpeMreB5 shows bundle formations, and its polymerization dynamics occur in the same manner as other actin superfamily members.

Evolution of polymer formation within the actin superfamily

While many are familiar with actin as a well-conserved component of the eukaryotic cytoskeleton, it is less often appreciated that actin is a member of a large superfamily of structurally related protein families found throughout the tree of life. Actin-related proteins include chaperones, carbohydrate kinases, and other enzymes, as well as a staggeringly diverse set of proteins that use the energy from ATP hydrolysis to form dynamic, linear polymers. Despite differing widely from one another in filament structure and dynamics, these polymers play important roles in ordering cell space in bacteria, archaea, and eukaryotes. It is not known whether these polymers descended from a single ancestral polymer or arose multiple times by convergent evolution from monomeric actin-like proteins. In this work, we provide an overview of the structures, dynamics, and functions of this diverse set. Then, using a phylogenetic analysis to examine actin evolution, we show that the actin-related protein families that form polymers are more closely related to one another than they are to other nonpolymerizing members of the actin superfamily. Thus all the known actin-like polymers are likely to be the descendants of a single, ancestral, polymer-forming actin-like protein.

Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 fromMethanopyrus kandleri

Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such asMethanopyrus kandlericontain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 fromM. kandleriin apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified.

Modeling studies of potato nucleoside triphosphate diphosphohydrolase NTPDase1: an insight into the catalytic mechanism.

Nucleoside triphosphate diphosphohydrolase--NTPDase1 (apyrase, EC 3.6.1.5) was modeled based on sequence homology. The single polypeptide chain of apyrase is folded into two domains. The putative catalytic site with the apyrase conserved regions (ACR 1-5) is located between these two domains. Modeling confirmed that apyrase belongs to the actin superfamily of proteins. The amino acids interacting with the nucleoside triphosphate substrate and probably involved in the catalyzed hydrolysis were identified. The proposed two-step catalytic mechanism of hydrolysis involves Thr127 and Thr55 as potential nucleophilic factors responsible for the cleavage of the Pgamma and Pbeta anhydride bonds, respectively. Their action seems to be assisted by Glu170 and Glu78 residues, respectively. The presence of two nucleophiles in the active site of apyrase explains the differences in the hydrolytic activity between apyrases and other enzymes belonging to the NTPDase family.

Urkinase: Structure of Acetate Kinase, a Member of the ASKHA Superfamily of Phosphotransferases

ABSTRACT Acetate kinase, an enzyme widely distributed in theBacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the α-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.