Purification and Properties of Two Isozymes of γ-Glutamyltranspeptidase from<i>Bacillus subtilis</i>TAM-4

Abe, Kazuhiro; Ito, Yoshihito; Ohmachi, Tetsuo; Asada, Yoshihiro

doi:10.1271/bbb.61.1621

Cited by 52 publications

(38 citation statements)

References 12 publications

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“…The ywtD product, however, has no amino acid sequence similarity to the CapD depolymerase, and it cleaves cPGA in vitro into fragments of 490 and 11 kDa (Suzuki & Tahara, 2003). B. subtilis GGT appears to be capable of generating Dand L-glutamate in vitro from cPGA (Abe et al, 1997). However, the precise hydrolytic mechanism of this enzyme has not been defined, and whether YwtD and GGT participate in the in vivo degradation process remains unknown.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Bacillus subtilis -glutamyltransferase and its involvement in the degradation of capsule poly- -glutamate

Kimura

2004

Microbiology

107

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During early stationary phase, Bacillus subtilis NAFM5 produces capsular poly(c-glutamic acid) (cPGA, 2610 6 Da), which contains D-and L-glutamate, and then degrades it during late stationary phase. The c-glutamyltransferase (EC 2.3.2.2; GGT) of this strain successively hydrolysed cPGA from the amino-terminal end, to yield both D-and L-glutamate. This enzyme was specifically synthesized during the stationary phase through transcriptional activation of the corresponding ggt gene by the ComQXPA quorum-sensing system. A ggt knockout mutant degraded cPGA into 1610 5 Da fragments, but not any further, indicating that the capsule cPGA is first internally degraded by an endo-type of cPGA hydrolase into 1610 5 Da intermediates, then externally into glutamates via GGT. Due to its inability to generate the glutamates from the capsule, the ggt mutant sporulated more frequently than the wild-type strain. The results show that B. subtilis GGT has a powerful exo-c-glutamyl hydrolase activity that participates in capsule cPGA degradation to supply stationary-phase cells with constituent glutamates.

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Section: Introductionmentioning

confidence: 99%

Characterization of Bacillus subtilis -glutamyltransferase and its involvement in the degradation of capsule poly- -glutamate

Kimura

2004

Microbiology

107

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“…Other PGA-degrading enzymes have been isolated from bacteria (1,10,25,28) and phage-infected cells of bacilli (7,29), including an endo-type enzyme purified from phage-infected cells and B. licheniformis (10), but the enzyme actions on PGA were not analyzed in detail. ␥-Glutamyltranspeptidase (GGT) from B. subtilis was reported to possess an exo-type PGA-hydrolyzing activity (1,18).…”

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confidence: 99%

“…␥-Glutamyltranspeptidase (GGT) from B. subtilis was reported to possess an exo-type PGA-hydrolyzing activity (1,18). Dep of B. anthracis, the gene for which is located immediately downstream of capBCA, similar to ywtD following ywsC and ywtABC, has also been shown to be a PGA-degrading enzyme, and the amino acid sequence exhibited high similarity with that of GGT (25).…”

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confidence: 99%

Characterization of the Bacillus subtilis ywtD Gene, Whose Product Is Involved in γ-Polyglutamic Acid Degradation

Suzuki

Tahara

2003

J Bacteriol

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The ywtD gene, which codes for an enzyme that degrades ␥-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the ␥-glutamyl bond only between D-and L-glutamic acids of PGA, and it may be designated ␥-DL-glutamyl hydrolase.␥-Polyglutamic acid (PGA), an amino acid polymer that consists of only D-glutamic acid or D-and L-glutamic acids polymerized through ␥-glutamyl bonds, is produced by several strains of Bacillus (4,11,19,21). The genes required for PGA biosynthesis have also been cloned as ywsC and ywtABC (capBCA or pgsBCA) from Bacillus anthracis (14) and Bacillus subtilis (2,26,27), and ywsC was found to code for PGA synthetase (EC 6.3.2), which is a crucial enzyme in PGA production, catalyzing the biosynthesis of PGA from L-glutamate in the presence of ATP and Mn 2ϩ ions (26). Sequencing of the complete genome of B. subtilis 168 (12) revealed that there is a gene (ywtD) located just downstream of ywsC and ywtABC that encodes a protein of 413 amino acids with a molecular mass of 45 kDa, and YwtD has been identified as an extracellular protein with a signal peptide of 32 amino acids, though its function remains unknown (6). Furthermore, a homology search revealed that a partial amino acid sequence of YwtD exhibits 40 and 37% identity with those of the catalytic domains of LytF and LytE, DL-endopeptidases that cleave the ␥-glutamyl bond between D-glutamic acid and L-diamino acid of cell wall peptidoglycan (15,20,22), suggesting that YwtD functions as an enzyme that degrades PGA (Fig. 1). In this study, we purified and characterized the YwtD protein, and we demonstrated that YwtD is a novel PGA-hydrolyzing enzyme that cleaves only the ␥-glutamyl bond between D-glutamic acid and L-glutamic acid of PGA.Construction of a plasmid containing ywtD with a histidinetag codon. Plasmid pYWTD for expression of ywtD from which a signal sequence (6) was excised was constructed as follows. A sense primer, 5Ј-CTCGAGGATATACATCAGAATTG-3Ј (XhoI site underlined), and an antisense primer, 5Ј-CTCGAG TTATTGGCACCCGTATACTTCC-3Ј (XhoI site underlined), were designed on the basis of the sequence of the ywtD gene of B. subtilis 168 (11). A DNA fragment was amplified by PCR with the two primers and B. subtilis IFO16449 (26) chromosomal DNA a...

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“…Among the bacterial c-GTs, those from Bacillus species have attracted interest [16][17][18][19], owing to some peculiar features.…”

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confidence: 99%

“…In this study, the donor and the acceptor substrates were used in the enzyme-catalyzed reactions in equimolar amounts, usually at concentrations of 100 mM. This approach gave us the opportunity to gain some insights into the enzyme activity, and to find new and unexpected behaviors with respect to those previously reported [16][17][18][19]40].…”

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confidence: 99%

pH‐Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ‐glutamyltransferase

et al. 2013

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c-Glutamyltransferases (c-GTs) are heterodimeric enzymes that catalyze the transfer of a c-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate c-glutamyl enzyme. In our search for a c-GT from a generally recognized as safe microorganism suitable for the production of c-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the c-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis c-GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis c-GT on pH, also in relation to the pK a of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four c-glutamyl moieties are linked to a single glutamine. Moreover, we found that D-glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis c-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of c-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material c-polyglutamic acid.

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Purification and Properties of Two Isozymes of γ-Glutamyltranspeptidase fromBacillus subtilisTAM-4

Cited by 52 publications

References 12 publications

Characterization of Bacillus subtilis -glutamyltransferase and its involvement in the degradation of capsule poly- -glutamate

Characterization of Bacillus subtilis -glutamyltransferase and its involvement in the degradation of capsule poly- -glutamate

Characterization of the Bacillus subtilis ywtD Gene, Whose Product Is Involved in γ-Polyglutamic Acid Degradation

pH‐Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ‐glutamyltransferase

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