Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A pneumococcal recombinant plasmid, pRG2, containing the lytA gene that codes for the pneumococcal N-acetylmuramoyl-L-alanine amidase has been constructed using the pneumococcal plasmid pLS1 as a vector. pRG2 was introduced by genetic transformation into a mutant of Streptococcus pneumoniae (M31) that has a complete deletion of the lytA gene. The transformed strain (M51) grew at a normal growth rate as 'diplo' cells and underwent autolysis at the end of the exponential phase of growth, two properties that had been lost in the deleted mutant M31. M51 lysed very rapidly at the end of the exponential phase when the cells were grown in choline-containing medium probably because of the higher level of amidase activity present in this strain as compared to the lysis-prone strain M11. These findings show that the expression of the plasmid-linked gene was placed under the mechanism(s) of control of the cell during the exponential phase. Our results demonstrate that the physiological role of the pneumococcal amidase was to catalyze the separation of the daughter cells at the end of the cell division to produce diplo cells; in addition we have also confirmed the basic role of this autolysin in the bacteriolytic nature of beta-lactam antibiotics.
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PMID:Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. 356 79

Autolysins are endogenous enzymes that specifically degrade the covalent bonds of the cell walls and eventually can induce bacterial lysis. One of the best-characterized autolysins, the major pneumococcal LytA amidase, has evolved by the fusion of two domains, the N-terminal catalytic domain and the C-terminal domain responsible for the binding to cell walls. The precise biochemical role played by the six repeat units that form the C-terminal domain of the LytA amidase has been investigated by producing serial deletions. Biochemical analyses of the truncated mutants revealed that the LytA amidase must contain at least four units to efficiently recognize the choline residues of pneumococcal cell walls. The loss of an additional unit dramatically reduces its hydrolytic activity as well as the binding affinity, suggesting that the catalytic efficiency of this enzyme can be considerably improved by keeping the protein attached to the cell wall substrate. Truncated proteins lacking one or two repeat units were more sensitive to the inhibition by free choline than the wild-type enzyme, whereas the N-terminal catalytic domain was insensitive to this inhibition. In addition, the truncated proteins were inhibited by deoxycholate (DOC), and the expression of a LytA amidase lacking the last 11 amino acids in Streptococcus pneumoniae M31, a strain having a deletion in the lytA gene, conferred to the cells an atypical phenotype (Lyt+ DOC-) (cells autolysed at the end of the stationary phase but were not sensitive to lysis induced by DOC), which has been previously observed in some clinical isolates of pneumococci. Our results are in agreement with the existence of several choline-binding sites and suggest that the stepwise acquisition of the repeat units and the tail could be considered an evolutionary advantage for the enzyme, since the presence of these motifs increases its hydrolytic activity.
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PMID:Carboxy-terminal deletion analysis of the major pneumococcal autolysin. 791 94

The genes hbl3, cpl1 and cpl7 coding for the pneumococcal phage lytic enzymes HBL3, CPL1 and CPL7, respectively, have been cloned into shuttle plasmids that can replicate in Streptococcus pneumoniae and Escherichia coli. All these genes were expressed in E. coli under the control of either the lytP promoter of the lytA gene, which codes for the major pneumococcal autolysin, or the promoter of the tetracycline-resistance gene (tetP). In contrast, cpl1 and cpl7 genes that code for lysozymes were expressed in pneumococcus only under the control of tetP, whereas the hbl3 gene that codes for an amidase can be expressed using either promoter. The phage lysozymes or amidase expressed in S. pneumoniae M31, a mutant deleted in the lytA gene coding for short chains, were placed under physiological control since these transformed bacteria grew as normal 'diplo' cells during the exponential phase and underwent autolysis only after long incubation at 37 degrees C. The lysis genes appear to be expressed constitutively in the transformed pneumococci, since sharply defined lysis of these cultures could be induced prematurely during the exponential phase of growth by addition of sodium deoxycholate.
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PMID:Lytic action of cloned pneumococcal phage lysis genes in Streptococcus pneumoniae. 847 29

The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in Escherichia coli. LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-beta-N-acetylglucosaminidase of Streptococcus pneumoniae. LytB can remove a maximum of only 25% of the radioactivity from [(3)H]choline-labeled pneumococcal cell walls in in vitro assays. Inactivation of the lytB gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence. Longer chains were formed when the lytB mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of lytA, which codes for the major autolysin. Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus. Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively. In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced. On the other hand, the use of a translational fusion between the gene (gfp) coding for the green fluorescent protein (GFP) and lytB supports the notion that LytB accumulates in the cell poles of either the wild-type R6, lytB mutants, or ethanolamine-containing cells (EA cells). The GFP-LytB fusion protein was also able to unchain the lytB mutants but not the EA cells. In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length. These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells.
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PMID:Purification and polar localization of pneumococcal LytB, a putative endo-beta-N-acetylglucosaminidase: the chain-dispersing murein hydrolase. 1219 14