The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtD EC , prtE EC , and prtF EC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtF EC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF EC system.The 62-kDa extracellular lipase of Serratia marcescens has no typical N-terminal signal sequence, but a sequence consisting of multiple repeats of nine amino acid residues (GGXGXD XXX), which is characterized as a glycine-and aspartic acidrich region, is situated in the C-terminal moiety. This sequence was found in the following extracellular proteins of gram-negative bacteria: metalloprotease from Erwinia chrysanthemi (5, 6); hemolysin, encoded by the hlyA gene in Escherichia coli (9); leukotoxin, encoded by the lktA gene in Pasteurella haemolytica (26); cyclolysin, a multifunctional protein carrying an adenylate cyclase activity and a hemolytic activity, encoded by the cyaA gene in Bordetella pertussis (11); and Ca 2ϩ -binding protein, encoded by the nodO gene in Rhizobium leguminosarum (7). The colicin V protein, the cvaC gene product of E. coli (10), possesses a repeated glycine-rich sequence which is not homologous to the GGXGXDXXX sequence but shares some characteristics with it. Since the E. coli cells carrying the S. marcescens lipA gene encoding the lipase did not secrete the lipase protein into the medium, the lipase is expected to be secreted extracellularly via a signal peptide-independent s...

SummaryThe Serratia marcescens Lip exporter belonging to the ATP-binding cassette (ABC) exporter is known to be involved in signal peptide-independent extracellular secretion of a lipase and a metalloprotease.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonasfluorescens B52 and P.fluorescens SIK Wi, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.Triacylglycerol acylhydrolase (EC 3.1.1.3) catalyzes the hydrolysis of triacylglycerol to glycerol and fatty acid and is generally called lipase. This enzyme is important in the food industry, and there is also current interest in the application of lipases to the production of chiral compounds. Extracellular lipases are produced by a variety of microorganisms: fungi, yeasts, and bacteria, including actinomycetes (60, 64). The lipase genes have been cloned from fungi (4, 21, 57, 58, 66), a yeast (32), Pseudomonas species (1, 6, 27, 29, 31, 34, 61a, 65), and staphylococci (19, 38). Serratia marcescens, an enteric bacterium, produces several extracellular enzymes which participate in the degradation of high-molecular-weight compounds, e.g., serine protease (67), chitinase (30), phospholipase (17), and nuclease (2, 7). Their genes have been cloned from S. marcescens, and the deduced amino acid sequences indicated that these proteins are secreted through a mechanism containing the secAY gene products with the aid of typical N-terminal signal peptides as described previously (51). Interestingly, the S. marcescens metalloprotease has been recently reported to be excreted through a mechanism similar to that of Escherichia coli ot-hemolysin having no N-terminal signal peptide (40).S. marcescens has been long known to produce extracellular lipase (64), but there is little knowledge of its enzyme. Very recently, we showed it to be stable in some organic solvents and to be applicable to the synthesis of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester by asymmetric hydrolysis of racemic trans-3-(4-methoxyphenyl)glycidic acid methyl ester, a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator (44). Our final goal is the construction of a lipase-hyperproducing strain of S. marcescens. This paper deals with the molecular cloning of the S. MATERIALS AND METHODSStrains and plasmids. E. coli K-12 DH5 (53) was used as a host for the construction of plasmids. The wild-typ...

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The gene for aromatic amino acid aminotransferase (ArAT) from Paracoccus denitrificans was cloned, sequenced, and overexpressed in Escherichia coli cells. The sequence differed from that reported previously [Takagi, T., Taniguchi, T., Yamamoto, Y., and Shibatani, T. (1991) Biotechnol. Appl. Biochem. 13, 112-119]. The enzyme (pdArAT) was purified to homogeneity, and characterized. It was similar to aspartate aminotransferase (AspAT) and ArAT of E. coli (ecArAT) in many respects, including gross protein structure and spectroscopic properties. pdArAT showed activities toward both dicarboxylic and aromatic substrates, and analysis of the binding of substrate analogs and quasisubstrates to the enzyme showed that both dicarboxylic and aromatic substrates take a similar orientation in the active site of pdArAT; these properties are essentially identical with those of ecArAT. As in the case of ecArAT, neutral amino acids with larger side chains are better substrates for pdArAT, suggesting that hydrophobic interaction between the substrate and the enzyme is important for the recognition of substrates with neutral side chains. pdArAT catalyzed transamination of phenylalanine and tyrosine far more efficiently (10(2)-fold in terms of kcat/Km) than those of straight-chain aliphatic amino acids with similar side-chain surface area, whereas ecArAT did not show significant preference for aromatic amino acids over aliphatic amino acids. This shows that the substrate-side-chain-binding pocket of pdArAT, as compared with the pocket of ecArAT, is well suited in shape for interaction with the phenyl and hydroxyphenyl rings of substrates. Thus, pdArAT is an ideal enzyme among ArATs for the study of the high-specificity recognition of two different kinds of substrates, the one having a carboxylic side chain and the other having an aromatic side chain.