Site-Directed Mutagenesis, Kinetic, and Spectroscopic Studies of the P-Loop Residues in a Low Molecular Weight Protein Tyrosine Phosphatase

Low molecular weight protein-tyrosine phosphatases (LMWPTPs) are small enzymes that ubiquitously exist in various organisms and play important roles in many biological processes. In Escherichia coli, the LMW-PTP Wzb dephosphorylates the autokinase Wzc, and the Wzc/Wzb pair regulates colanic acid production. However, the substrate recognition mechanism of Wzb is still poorly understood thus far. To elucidate the molecular basis of the catalytic mechanism, we have determined the solution structure of Wzb at high resolution by NMR spectroscopy. The Wzb structure highly resembles that of the typical LMW-PTP fold, suggesting that Wzb may adopt a similar catalytic mechanism with other LMW-PTPs. Nevertheless, in comparison with eukaryotic LMW-PTPs, the absence of an aromatic amino acid at the bottom of the active site significantly alters the molecular surface and implicates Wzb may adopt a novel substrate recognition mechanism. Furthermore, a structure-based multiple sequence alignment suggests that a class of the prokaryotic LMW-PTPs may share a similar substrate recognition mechanism with Wzb. The current studies provide the structural basis for rational drug design against the pathogenic bacteria.Low molecular weight protein-tyrosine phosphatases (LMW-PTPs) 3 are small cytoplasmic enzymes (ϳ18 kDa) that are widely distributed in prokaryotes and eukaryotes (1, 2). In eukaryotes, LMW-PTPs specifically dephosphorylate and down-regulate many tyrosine kinase receptors, such as platelet-derived growth factor receptor (3, 4), insulin receptor (5), or ephrin receptor (6). The reaction mechanism of eukaryotic LMW-PTPs has been structurally, thermodynamically, and kinetically characterized (7). In contrast, very limited knowledge is available for prokaryotic LMW-PTPs thus far. All LMW-PTPs share a highly conserved C(X) 5 RS motif, where X can be any amino acid. This motif adopts a loop structure, where the phosphate group of the substrate binds to, and is known as the P-loop (1). In addition, some amino acids required for the catalysis are also highly conserved, such as an aspartate residue that acts as a general acid (8). Based on the structures of mammalian LMW-PTPs in complex with exogenous substrates or serendipitous ligands, determinants for substrate specificity were proposed, including the ring stacking around the targeted phosphotyrosine provided by two aromatic side chains of the LMW-PTPs (9, 10).Many bacterial species harbor a layer of capsular polysaccharides and surface-associated exopolysaccharides (EPS). In Escherichia coli, the group 1 capsular polysaccharides and the colanic acid EPS are assembled in a Wzy-dependent polymerization system (11). The ca gene cluster contains one tyrosine autokinase (Wzc) and one LMW-PTP (Wzb) that function as a pair of kinase/phosphatase in the regulation of colanic acid production (12, 13). As a consequence, colanic acid is only produced after the dephosphorylation of the phosphorylated Wzc by Wzb (14). Wzc was identified to regulate the activity of UDPglucose dehydrogenase b...

Section: Discussionmentioning

confidence: 99%

“…This may explain why Wzb is inactive on phosphoserine or phosphothreonine (13). In addition, six charged residues (Lys 43 , Glu…”

Section: Discussionmentioning

confidence: 99%

The Solution Structure of Escherichia coli Wzb Reveals a Novel Substrate Recognition Mechanism of Prokaryotic Low Molecular Weight Protein-tyrosine Phosphatases

Lescop

Hu²,

Xu³

et al. 2006

“…Thus, the hydroxyl may play a relatively greater role the first step of BVP catalysis than it does in the case of protein-tyrosine phosphatases and dual specificity protein phosphatases. BVP appears similar in its utilization of the catalytic threonine to the mammalian low molecular weight protein-tyrosine phosphatase, in which the equivalent serine is proposed to facilitate attack of the cysteine thiolate on the scissile phosphate (38 ) in a manner that is specific to the RNA triphosphatase subfamily of cysteinyl phosphatases.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Phosphoanhydride Cleavage by Baculovirus Phosphatase

Martins

Shuman

2000

Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus. BVP and other metazoan RNA triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a covalent cysteinyl-phosphate intermediate. ) invoked as a candidate general acid catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP. We propose that the low pK a of the bridging oxygen of the ␤ phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the ␥ phosphorus. In contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving groups have a much higher pK a than does ADP.RNA triphosphatase catalyzes the hydrolysis of the ␥ phosphate of triphosphate-terminated RNA to form a diphosphate end. Two distinct classes of eukaryotic RNA triphosphatases have been described. The RNA triphosphatases of metazoans and plants belong to a superfamily of phosphatases that includes protein-tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases (1-12). The metazoan RNA triphosphatases do not require a metal cofactor. In contrast, the RNA triphosphatases of fungi and DNA viruses (poxviruses and baculoviruses), which comprise a new family of nucleoside triphosphate phosphohydrolases, are strictly dependent on a divalent cation cofactor (13-19). There are no mechanistic or structural similarities whatsoever between the metazoan and viral/fungal triphosphatase families (20). Thus, RNA triphosphatase presents a remarkable case of complete divergence during the transition from fungal to metazoan species.The metazoan RNA triphosphatases are subdivided into two groups: (i) bifunctional enzymes composed of an N-terminal RNA triphosphatase domain and a C-terminal RNA guanylyltransferase domain (1-9) and (ii) monofunctional RNA triphosphatases (10 -12). The bifunctional enzymes are ubiquitous in higher eukaryotes, where they are responsible for forming the 5Ј cap structure of cellular mRNAs. The monofunctional enzymes perform RNA 5Ј modification reactions uncoupled from or unrelated to cap formation, and their true role in RNA metabolism is not understood. The latter category includes BVP, 1 a phosphatase encoded by the Autographa californica baculovirus (10, 11), and the human PIR1 phosphatase (12). Also in the latter category are the alternatively spliced forms of the cellular capping enzymes that retain the RNA triphosphatase domain but lack essential constituents of the guanylyltransferase active site (7-9).The RNA triphosphatase domains of the metazoan and plant capping enzymes contain the signature HCXXGXXR(S/T) phosphate-binding motif of the protein-tyrosine phosphatase/dual specificity protein phosphatase enzyme superfamily (Fig. 1). Protein-tyrosine phosphatases and dual specificity protein phosphatases cat...

“…Recently, VHR has been identified as a tyrosine-specific extracellular signalregulated kinase phosphatase (16). Like other PTPs the activesite Cys-124 is stabilized by an extensive network of hydrogen bonding between backbone NH of the P-loop and the thiolate anion (4,17,18). The sulfhydryl group of Cys-124 has an unusually low pK a value and functions as a nucleophile to form a thiophosphate-enzyme intermediate (19).…”

mentioning

confidence: 99%

“…The water molecule ligated at this position seems to be unique to VHR. His-123 of VHR is known to be conserved in most PTPs as HC(X) 5 R(S/T) except low molecular weight PTPs where His is replaced by Val (17). However, studies on the function of the conserved His are surprisingly sparse.…”

mentioning

confidence: 99%

Mutational and Kinetic Evaluation of Conserved His-123 in Dual Specificity Protein-tyrosine Phosphatase Vaccinia H1-related Phosphatase

Kim

Shin²,

Han³

et al. 2001

Active-site cysteine strategically positioned in the Ploop of protein-tyrosine phosphatases has been suggested to be further stabilized by hydrogen bonding arrays radiating out from the P-loop to neighboring residues. In this work, we investigated the structural role of histidine array in HC(X) 5 RS motif of the vaccinia H1-related protein phosphatase (VHR), using site-directed mutagenesis in conjunction with an extensive kinetic analysis. Conserved His-123 was mutated along with neighboring residues Tyr-78 and Thr-73. The increased pK a values of active-site Cys-124 found in Y78F and T73A mutants (6.51 and 6.75, respectively) were comparable to those of H123A and H123F mutants. Kinetic evaluation of Y78F and T73A mutants further implicates that the mutations perturb the relative position of Cys-124 within the P-loop. These results imply that Tyr-78 and Thr-73 make up an essential part of the His-123 array and structurally tune the Cys-124 position. Tyr-78 of VHR turns out to be the invariant Tyr reported in several protein-tyrosine phosphatases by a structurebased sequence alignment. Therefore, orientation of the imidazole ring of His-123 by the invariant Tyr-78 is crucial for maintaining the proper position of Cys-124 in the P-loop.The superfamily of protein-tyrosine phosphatases (PTPs)