[42] Phospholipase B from Penicillium notatum

Saito, Kunihiko; Sugatani, Junko; Okumura, Tadayoshi

doi:10.1016/0076-6879(91)97170-4

Cited by 41 publications

(53 citation statements)

References 22 publications

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“…The large and small peptides of the modified enzyme were produced by proteolytic processing between Leu175 and Asp176, and consisted of428 and 175 amino acids with calculated molecular masses of46642 Da and 18 155 Da, respectively. These values were comparable with those determined by SDSjPAGE [5].…”

Section: Isolation C?f'cdna Clone Und D N a Sequence Anulysissupporting

confidence: 78%

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Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA

Masuda

Kitamura

Saito

1991

European Journal of Biochemistry

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Phospholipase B has not yet been well defined. The most important points about this enzyme are its relationships with lysophospholipase and phospholipase Al. As reported [Saito, K., Sugatani, J. & Okumura, T. (1991) Mc>thod,s Enzymol. 197, 446 -4561, Penicillium notutum phospholipase B is a glycoprotein with a molecular mass of 95 kDa and intrinsic lysophospholipase and phospholipase B activities; however, by endogenous proteolytic modification, its phospholipase B activity is lost almost completely, whereas its lysophospholipase activity remains unchanged.A cDNA library of P. notaturn was screened by hybridization with two synthetic oligodeoxyribonucleotide probes, which corresponds to two different pentapeptides of the enzyme. A hybridization-positive clone, pPLBl8, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence was quite different from that found previously. Therefore, we rescreened the cDNA hbrdry with a Suu3AI fragment derived from pPLB18 and isolated a new clone, pPLB15. Comparison of the nucleotide sequences of pPLB15 and pPLB18 revealed that pPLB18 contained an insertion sequence of 53 bp. Consequently, the reading frame was open downstream for 603 amino acid residues.From the assigned sequence, it was deduced that the limited proteolysis occurred between Leu175 and Asp176; eight cysteine residues and 16 potential N-glycosylation sites were also found. No amino acid sequence similarity was found with other proteins, including other phospholipases.Phospholipase B has been found in microorganisms, plants and animal tissues; in many cases it has lysophospholipase activity in addition to phospholipase B activity, as reviewed by van Penicilliurn notutum phospholipase B is a glycoprotein with a molecular mass of about 95 kDa and intrinsic phospholipase B and lysophospholipase activities. However, on proteolytic modification with an endogenous protease(s), phospholipase B activity was almost completely lost, whereas lysophospholipase activity was unaffected [5].In this paper, we describe the isolation of a cDNA for the phospholipase B from P. notaturn and assign the primary structure of the protein moiety of this enzyme. The primary structure is discussed in relation to the enzymatic properties reported before [5, 61, and also to lysophospholipase and phospholipase A l .

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Section: Isolation C?f'cdna Clone Und D N a Sequence Anulysissupporting

confidence: 78%

“…However, on proteolytic modification with an endogenous protease(s), phospholipase B activity was almost completely lost, whereas lysophospholipase activity was unaffected [5].…”

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confidence: 99%

Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA

Masuda

Kitamura

Saito

1991

European Journal of Biochemistry

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“…In contrast to PLB from the relatively non-pathogenic fungus, Penicilliurn notaturn -where acidic, inner leaflet phospholipids PS, PI, PA are preferred substrates [24] -cryptococcal PLB showed greatest activity with DPPC, followed by PG. These are the two major phospholipid components of lung surfactant [26], and the finding that bovine lung surfactant was degraded at a rate similar to DPPC supports the hypothesis that cryptococcal PLB assists the organism to penetrate the surfactant lining of the alveolar epithelium.…”

Section: Discussionmentioning

confidence: 99%

“…Strain BL-1 was grown to confluence on SDA for 72 h at 30"C, washed and resuspended in harvesting buffer (10 mM imidazole, 2 M CaC12, 2 mM MgC12, 56mM D-glucose, in NaCl 0.9% w/v, pH 5.5) for incubation (20)(21)(22)(23)(24) h at 37°C). The cell-free supernate was assayed for total protein content and stored at -70°C until required.…”

Section: Preparation Of Cryptococcal Supernates Containing Extracellumentioning

confidence: 99%

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Santangelo

Nouri-Sorkhabi

Sorrell

et al. 1999

Journal of Medical Microbiology

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A recent study demonstrated that phospholipase B (PLB), lysophospholipase (LPL) and lysophopholipase transacylase (LPTA) are secreted by Cryptococcus neoformans var. neoformans and showed that the amount of enzyme production correlated with virulence in mice. The present study characterised the extracellular enzyme activities further by radiometric assays and 31 P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40OC. Bovine lung surfactant and its major lipid components, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB. Lysophosphatidylcholine was the favoured substrate for LPL and LPTA. PLB and LPL/LPTA were differentially affected by Triton X-100, and palmitoyl carnitine was a potent inhibitor of the three phospholipases. LPL and PLB activities were inhibited by dithiothreitol; N-ethylmaleimide inhibited LPL and LPTA activities. None of the enzymes was inhibited by N-bromosuccinimide or p-bromophenacyl bromide. Cellular disruption experiments indicated that > 85% of the phospholipase activities were cell-associated, with LPL and LPTA being more easily released than PLB. At pH 5.5 and 7.0, the heat-inactivated secreted enzyme preparations decreased the viability of human neutrophils. This effect was attenuated by active supernates. The relative activities of the PLB, LPL and LPTA in the environment of neutrophils are likely to determine the fate of these cells in vivo. Both phospholipases and heat-stable substances secreted by C. neoformans at 37°C could contribute to membrane degradation and virulence.

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“…Additionally, lysophospholipase-transacylase activity is associated with some PLB enzymes, allowing these enzymes to transfer a FFA to a lysophospholipid and produce a diacylphospholipid. Hydrolase and acyltransferase activities have been detected in several fungi, including Saccharomyces cerevisiae [5], Candida albicans [6], Candida utilis [7], Penicillium chrysogenum [8] and Cryptococcus neoformans [9]. PLBs from Escherichia coli [10] and Bacillus subtilis [11] are known; however, there has been no investigation of bacterial PLBs with respect to whether they produce lysophospholipids during diacylglycerophospholipid hydrolysis and possess lysophospholipase-transacylase activity.…”

Section: Introductionmentioning

confidence: 99%

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

et al. 2013

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A novel metal ion-independent phospholipase B (PLB 684 ) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 UÁmg protein À1 ). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50°C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB 684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent K m , V max and k cat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 mM, 15.8 mmolÁmin À1 Ámg protein À1 and 1.02 9 10 4 s À1 , respectively. The positional specificity of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1 : sn-2) was 45 : 55. The ORF of the gene is 1239 bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. The deduced amino acid sequence of PLB 684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0). The extracellular production of PLB 684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB 684 and that the active site may include residues Ser330 and His332.

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[42] Phospholipase B from Penicillium notatum

Cited by 41 publications

References 22 publications

Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA

Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

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