biosynthesis in Klebsiella pneumoniae CG43
Tyrosine phosphorylation of Ugd is catalyzed by the cytoplasmic PTK domain of Yco6. Although the tyrosine phosphorylation regulated by PTK and PTP has been shown to modulate biosynthesis and assembly of CPS (Vincent et al., 2000), the mechanism is still unknown. Since the recent reports suggested that E. coli Ugd and B.
subtilits Ugd are the substrates for PTK (Grangeasse et al., 2003; Mijakovic et al., 2003), we herein intend to demonstrate in K. pneumoniae CG43 that the Ugd could also be phosphorylated by the PTK, Yco6. In comparing the amino acid sequences of K. pneumoniae Ugd (KpUgd) (NTUH-K2044 strain, NCBI accession No. BAD03946) with E. coli Ugd (K12 strain, NCBI accession No. NP_416532) and B. subtilis YwqF (strain 168, NCBI accession No. CAB15640), approximately 83% and 26% sequence identities were observed respectively. Interestingly, 99.2% amino acid sequence identity was found between KpUgd and the Ugd of E. coli K30 strain (NCBI accession No. AAC45348) indicating they are more related. To demonstrate if a similar control of Ugd by PTK Yco6 is present in K. pneumoniae, the purified His6-KpUgd was incubated with the phosphorylated Yco6, His6-Yco6 (Arg451-Lys722), and [γ-32P] ATP. As shown in Fig. 12A, Yco6 could auto-phosphorylate itself in the presence of [γ-32P] ATP (lane 5) and the phosphorylation of KpUgd could be detected (lane 6) by comparing to the non-radioisotope-labeled His6-KpUgd while the protein incubated with only [γ-32P] ATP (lane 1). While incubating with the phosphorylated His6-Wzc (Ser447-Ala704), the PTK of E. coli and deleted from its tyrosine cluster, in the presence of [γ-32P] ATP, the His -KpUgd appeared to be also phosphorylated (Fig.
12B, lane 2), suggesting that the two PTK, Yco6 and Wzc, exert a similar mechanism via tyrosine phosphorylation to modulate the activity of Ugd.
Phosphorylation enhances the Ugd activity. Ugd is UDP-glucose dehydrogenase catalyzing a NAD+-dependent transformation of UDP-glucose into UDP-glucuronic acid (Pagni et al., 1999). Phosphorylation of the UDP-glucose dehydrogenases in E.
coli and B. subtilis enhancing their enzymatic activities have recently been reported (Grangeasse et al., 2003; Mijakovic et al., 2003). Consistent with the findings, the enzymatic activity could be enhanced when the recombinant KpUgd was incubated with His6-Yco6(Arg451-Lys722) and ATP (Fig. 13). When KpUgd was incubated with His6-Wzc(Ser447-Ala704) and ATP, its activity was also enhanced. In addition, incubation of the phosphorylated His6-KpUgd with calf intestine alkaline phosphatase (CIAP) (Fermentas) dramatically reduced the Ugd activity, which indicating that phosphorylation of Ugd indeed enhanced its activity, and the purified His6-KpUgd contained a small fraction of the phosphorylated form.
Tyrosine phosphorylation of either of Gnd, ManC and ManB could be catalyzed by Yco6 and Wzc. In the biosynthesis of E. coli group 1 CPS, there are several glycosyltransferases and enzymes, including ManBC and Ugd, involved in the synthesis of sugar nucleotide precursors (Whitfield and Paiment, 2003). Besides, Gnd, a 6-phosphogluconic dehydrogenase, is also required for the formation of the cellular polysaccharide layer (Sprenger, 1995). As shown in Fig. 1, ManBC and Gnd are encoded by the genes in the cps gene cluster of E. coli and K. pneumoniae. The possibility of ManBC and Gnd are also regulated by tyrosine phosphorylation was investigated. When the purified His6-Gnd, His6-ManC and His6-ManB were incubated respectively with [γ-32P] ATP and the phosphorylated His6-Yco6(Arg451-Lys722), phosphorylations of these proteins were all detected (Fig. 12A, lane 8, 10 and 12)
comparing to those when the proteins incubated only with [γ-32P] ATP (Fig. 12A, lane 2, 3 and 4). When the purified His6-Gnd, His6-ManC and His6-ManB were incubated respectively with [γ-32P] ATP and the phosphorylated His6-Wzc(Ser447-Ala704), the phosphorylation signals of these proteins were also observed (Fig. 12B, lane 3, 4 and 5). This indicated that phosphorylations of these proteins did not require the tyrosine cluster of PTK.
Phosphorylation of Gnd enhances its 6-phosphogluconic dehydrogenase activity. Gnd is a 6-phosphogluconic dehydrogenase, which catalyzes the oxidative decarboxylation of 6-phospho-gluconate to ribulose-5-phosphate (Sprenger, 1995).
When the purified His6-Gnd was incubated with ATP and either His6-Yco6(Arg451-Lys722) or His6-Wzc(Ser447- Ala704), increasing enzymatic activity was observed (Fig. 14). Incubation of the His6-Gnd with CIAP (Fermentas) also appeared to reduce its activity indicating that the purified His6-Gnd contained some of the phosphorylated form.
The phosphorylated form of Ugd, Gnd, and ManC could be modified by Yor5.
In general, bacteria most often contain antagonistic PTKs and PTPs, and the genes encoding PTKs and PTPs are located next to each other in the same gene clusters. In bacteria, PTK and PTP have been reported to play important roles in regulation of the CPS and EPS production (Nakar and Gutnick, 2003; Stevenson et al., 1996). Many of the PTPs could specifically dephosphorylate the cognate PTKs and also the substrates of PTK (Grangeasse et al., 1998; Mijakovic et al., 2005; Morona et al., 2002;
Musumeci et al., 2005; Vincent et al., 1999). To demonstrate if the phosphorylations demonstrated above could be modified specifically by Yor5, aliquots of the purified His6-Yor5 were added respectively into the above phosphorylation mixtures and incubated at 37℃ for 30 min. As shown in Fig. 12A, the radioactive labels of Gnd
(lane 9) and ManC (lane 11) were removed. On the contrary, no effect on the phosphorylated ManB (lane 13) was found. Interestingly, the phsophorylated KpUgd appeared to be a poor substrate for the recombinant Yor5 (Fig. 12A, lane 7).
Moreover, significant amount of Yco6 and Wzc remained to be phosphorylated (Fig.
12A, lane 7, 9, 11, 13, and Fig. 12B, lane 6). I speculate that Yor5 could efficiently remove the phosphorylated tyrosine residues at the C-terminal tyrosine cluster of Yco6. However, the modification at Tyr570 which is related to the kinase activity of Yco6 could be low. Therefore, Yor5 would regulate the activity of KpUgd, Gnd, ManC and Yco6 by desphosphorylation. On the other hand, ManB is likely regulated by the other phosphatase.
Discussion
The presence of homologues of Wzi, Wza, Yor5, Yco6, Wzx, and Wzy in K.
pneumoniae raises the question of whether these proteins are functional conserved.
The gene, wzi, is only found in the cps loci of K. pneumoniae and E. coli group 1 CPS serotype. Its role in linking high-molecular-weight CPS to cell surface has been demonstrated recently (Alvarez et al., 2000; Rahn et al., 2003). Although the precise way of the linkage is not clear, it is noticeable that wzi is confined to the bacteria wherein the capsule is a major virulence factor and their CPS polymers are tightly linked to the cell surface. Wza is assumed to be a multimeric outer membrane protein complex required for surface expression of CPS (Nesper et al., 2003), and wza -mutation in K. pneumoniae also showed apparent loss of surface CPS in this study.
The block of translocation of polymeric CPS also resulted in reduction of total CPS biosynthesis. The block of translocation of CPS polymer has been suggested to feedback inhibit the upstream CPS biosynthesis, but the exact mechanism remains to be determined (Nesper et al., 2003). As shown in Fig. 6, expression of Wza in wza -mutant could only partially restore the phenotype might result from an inappropriate amount of the expressed protein. Nevertheless, three-dimensional structure of Wza has recently been resolved, which indicated that Wza is an octameric complex with a tetrameric (C4) rotational symmetry and is organized as a tetramer of dimeric subunits (Beis et al., 2004).
Compared with Wza, the studies of Wzx and Wzy are less and confined in O-antigen synthesis, due to the difficulties of purification of the membrane protein and verification of their activities. Some polymeric CPS found in wzx- mutant (Fig. 8) indicated that oligomer could be flipped across inner membrane without Wzx, and
Wzx might not be the only translocator required in the process. The amino sequence of Wzy is variable among strains (identities are less than 16% among K. pneumoniae Chedid and NTUH K2044, E. coli K12 and K30), and this implies that it is specific to each serotype. So far, no Wzy homolog has been purified and studied at biochemical level to confirm its polymerase activity. In E. coli serotype K30 (Drummelsmith and Whitfield, 1999) and K40 (Amor and Whitfield, 1997), wzy mutants lack capsules and add only a single K antigen repeat unit to the LPS lipid A core, namely KLPS, implying the role of Wzy for CPS polymerization. Both yco6 and yor5 mutants lost high-molecular-weight CPS, suggesting that Yco6 and Yor5 are involved in polymerization of CPS. Overall, mutation in either of the core elements exerted capsule-lacking phenotype. Using the assays of polymyxin B sensitivity and biofilm formation, yor5 and wzx mutants were found to be more susceptible to polymyxin B and increase capability of biofilm formation. Nevertheless, the mechanism is still a question to be investigated.
After thorough analyses, it is believed that PTKs are involved in the regulation of CPS or EPS production (Vincent et al., 2000). The finding that UDP-glucose dehydrogenases are phosphorylated by PTKs strongly supports this viewpoint, although PTKs might play other roles via other mechanisms (Klein et al., 2003;
Mijakovic et al., 2006). Herein, we have demonstrated that, in addition to Ugd, Gnd and ManBC are also regulated by tyrosine phosphorylation via Yco6. The phosphorylation appeared also to increase the enzymatic activity, respectively.
Although the effect of phosphorylation on ManBC activity has not yet been verified, enhancement of the activity is predictable. Through dephosphorylation by Yor5, the phosphorylated residues is at tyrosine could be inferred. To identify the phosphorylated residue, the amino acid sequences of the enzymes, KpUgd, Gnd and
ManBC, and known substrates of PTK, Ugd and Ssb, were comparatively analyzed.
There are 14 to 25 tyrosine residues existing in each protein, however, no conserved motif was found. The phosphorylated site may be determined according to the three-dimensional structure of protein substrate but not specific primary amino acid sequence. In recently, mass spectrometry allowed to pinpoint the phosphorylated tyrosine residue in B. subtilis Ssb (Mijakovic et al., 2006), and the phosphorylated residues of KpUgd, Gnd and ManBC might be analyzed by the same method.
Besides the enzymes for CPS precursor synthesis, it is known that the PTKs also autophosphorylate themselves and the phosphorylations at C-terminal tyrosine residues seem to be unrelated to activations of their substrates (Grangeasse et al., 2003). Their kinase activity appeared to be relative to the upstream tyrosine residue and Walker A and B motifs. Moreover, the phosphorylation of the upstream tyrosine and the substrates could not or slowly be dephosphorylated by their cognate PTPs.
Only the tyrosine residues in the C-terminal tyrosine cluster of PTKs could be entirely modified by the cognate PTPs implying that different PTP existed to exert the activity to dephosphorylate the upstream tyrosine residue and the tyrosine of certain substrates.
The three-dimensional structure of Wzc (Collins et al., 2006) and the finding of the Wza-Wzc interaction (Nesper et al., 2003) indicated that Wzc, as a tetrameric complex with C4 rotational symmetry, forms multienzyme complex with Wza and other membrane proteins which is involved in polymerization and translocation of CPS. The overall level of phosphorylation in the tyrosine cluster is important for capsule assembly, but not the upstream tyrosine (Paiment et al., 2002). Briefly, the PTP-modifiable phosphorylations on the C-terminal tyrosine clusters of PTKs are probably related to conformational changes of PTKs which result in modulating conformation of the multienzyme complex to open or close the translocation channel
of CPS.
As shown in Fig. 15, we propose a model which predicts that Yco6 would regulate both CPS biosynthesis and translocation by phosphorylation. The PTK activity of Yco6 is first enhanced by autophosphorylation at Tyr and the resulting activation of Yco6 stimulates interphosphorylation at C-terminal tyrosine cluster and also the phosphorylations of its substrates. The phosphorylations of these substrates increase their activities resulting in elevated synthesis of sugar nucleotide precursors. CPS lipid-linked repeat unit composed of sugar nucleotide precursors then is flipped across the inner membrane in a process requiring Wzx and polymerized through Wzy-dependent reaction. Finally, CPS polymer is exported to cell surface via the channel formed by Wza and associated on the surface per the outer membrane protein, Wzi. Although Yco6 also plays roles in CPS polymerization and translocation, it is not yet clear whether these activities require the phosphorylation of Yco6 at specific tyrosine in the C-terminal tyrosine cluster.
570
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