Lyase cloning

Chung, B., J. Picado-Leonard, M. Haniu, M. Bienkowski, RE Hall, J.E. Shively etal. (1987). Cytochrome P450c 17 (steroid 17a-hydroxylase/17, 20 lyase) Cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. Proc. Natl. Acad. Sci. USA 84,407-411. 

The chemoenzymatic synthesis of (13S)-hydroxy-18 2(9Z,ll ) was achieved in nine steps starting from (2 )-octenal. Of importance was the enzymatic conversion of (2 )-octenal to the (5)-cyanohydrin [5] with (S)-hydroxynitrile lyase cloned from Hevea brasiliensis (41). 13- Hydroxy-lO-oxo-18 1(11 ) was synthesized via a Kno-evenagel-type reaction of Isopropyl 11 - phenylsulfinyl-10-oxoundecanoate with hep-tanal to form y-hydroxyenone functionality together with carbon chain elongation (42). The regiospecific oxidation of a number of substituted unsaturated fatty esters with /7-benzoquinone in the presence of palladium(II) chloride under concomitant ultrasonic irradiation was reported. For example, methyl 9-hydroxy-18 1 (12Z) furnished methyl 9-hydroxy-12-keto-18 0 exclusively (43). 

Cloning and heterologous expression of icapectete lyase genes... 

In order to characterize the pectinolytic enzymes encoded by these clones, the culture supernatants of all these clones were tested for pectate lyase and polygalacturonase activity, using thiobarbutiric acid as described in materials and methods. Absorption at 550 nm indicates the activity of pectate lyase whereas absorption at 510 nm indicates the activity of polygalacturonase. 

We have subcloned the coRI restriction fragment of 9.2 kbp from the clone PFAJ601 in pLAFRl, and as expected, this fragment is sufficient to express pectate lyase and polygalacturonase activity in E. coli tranconjugants... 

Chen C-M, Q-Z Zhuang, Z Zhu, BL Wanner, CT Walsh (1990) Molecuar biology of carbon-phosphorus bond cleavage Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli. J Biol Chem 265 4461-4471. 

Hasslacher, M., Schall, M., Hayn, M. et al. (1996) Molecular cloning of the full-length cDNA of (5)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue. The Journal of Biological Chemistry, 271, 5884-5891. 

This pyridoxal phosphate-requiring enzyme has been studied in several bacteria and X-ray crystal structures are available.35 The coryneform bacterium, Brevi-bacterium linens, is common on the surface of several cheeses, including Limburger and those of the Trappist type. The methionine y-lyase of this organism has been purified to homogeneity36 and the relevant gene, mgl (from MGL, abbreviation for methionine y-lyase) has been cloned and analyzed.37... 

MATSUI, K., WILKINSON, J., HIATT, B., KNAUF, V., KAJIWARA, T., Molecular cloning and expression of Arabidopsis fatty acid hydroperoxide lyase, Plant Cell Physiol., 1999,40,477-481. 

The intracellular concentrations of sphingosine and SIP are governed by the activities of enzymes that catalyse their synthesis and removal. These include ceramidase, sphingosine kinase (SPHK), SIP phosphatase and SIP lyase (Figure 1). Several of these enzymes have only recently been cloned and knowledge of their respective roles and regulation is incomplete. 

Erwinia chrysanthemi, interest in the maceration process has led to the cloning and sequencing of four separate genes coding for different secreted pectate lyases (18,19), The production of these enzymes, either by the parent organism, or as recombinant enzymes in E. coli, has now led to their detailed characterization with respect to depolymerization mechanism. 

ACC 71 synthase, i. e. (S)-adenosylmethionine methylthioadenosine lyase (EC 4.4.1.14), has been purified from several plant tissues [116]. Recently, ACC synthase cDNA clones have been isolated and sequenced from wounded fruit tissues of tomato, winter squash, zucchini, ripening apple and tomato fruit. Using the polymerase chain reaction (PCR), four different ACC synthase gene fragments were obtained by amplification of cDNA derived from mRNA of tomato... 

Several biotransformation processes were developed. As an example Allelix used screening to isolate a Corymbacterium capable of converting df-acetamidocirmamic acid into L-phenylalanine. Lactate was added to the media to stimulate NADH regeneration. The key enzymes involved were cloned to increase copy numbers, the cells were used in iimnobilized form and phenylalanine recovery was facilitated by precipitation techniques. This approach was also explored by the Tanabe Co. in Japan. By contrast Cenex used the phenylalanine arrrmonia lyase activity of Rhodococcus rubra to convert irans-cinnamic acid. 

The detailed characterization of hydroxynitrile lyases from Sorghum hicolor (E.C. 4.1.2.11) and Linum usitassimum (E.C. 4.1.2.37) has been hampered for a long time due to the lack of a recombinant expression system. Therefore our studies were focused on cloning of the coding genes, recombinant expression, and characterization of these enzymes. 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and... 

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