- Email: [email protected]
Cloning, sequencing and overexpression of the gene encoding malate dehydrogenase from the deep-sea bacterium Photobacterium species strain SS91
Biochimica et Biophysica Acta 1350 Ž1997. 41–46
Short sequence-paper
Cloning, sequencing and overexpression of the gene encoding malate dehydrogenase from the deep-sea bacterium Photobacterium species strain SS9 1 Timothy J. Welch, Douglas H. Bartlett
)
Center for Marine Biotechnology and Biomedicine, Marine Biology Research DiÕision, Scripps Institution of Oceanography, UniÕersity of California, 9500 Gilman DriÕe, San Diego, La Jolla, CA 92093-0202, USA Received 25 July 1996; revised 10 October 1996; accepted 14 October 1996
Abstract The gene encoding malate dehydrogenase Ž mdhA. was obtained from the psychrophilic, barophilic, deep-sea isolate Photobacterium species strain SS9. The SS9 mdhA gene directed high levels of malate dehydrogenase ŽMDH. production in Escherichia coli. A comparison of SS9 MDH to three mesophile MDHs, a MDH sequence obtained from another deep-sea bacterium, and to other psychrophile proteins is presented. Keywords: Psychrophile; Barophile; Cold adaptation; High pressure adaptation; TCA cycle; Malate dehydrogenase; Ž Photobacterium.; Ž E. coli .
Malate dehydrogenase ŽMDH; L-malate-nicotine adenine dinucleotide wNADx oxidoreductase, EC 1.1.1.37., catalyzes the NADŽH. -dependent interconversion of malate and oxaloacetate. Although MDHs exist in different isozyme forms, and possess roles in diverse metabolic activities including aspartate biosynthesis, the malate-aspartate shuttle, gluconeogenesis, and lipogenesis, MDH is best known as an enzyme of the tricarboxylic acid cycle w1x. Along with lactate dehydrogenase Ž LDH. , another 2-hydroxy acid dehydrogenase, as well as other dehydrogenases, MDH has been one of the premier enzymes used for examining protein biochemical adaptation to
)
Corresponding author. Fax: q1 Ž619. 5347313; E-mail: [email protected] 1 The sequence data reported in this paper is present in the GenBankr EMBL libraries under the accession number L13319.
extreme environments. For example, kinetic information exists for MDHs from a variety of mesophilic, thermophilic, and halophilic microorganisms Žexamples in Refs. w2–6x.. Numerous malate dehydrogenase gene sequences and protein sequences are also available from Eucarya w7–12x, Archaea w13,14x, and Bacteria w17–20x. The amino-acid sequences of tricarboxylic acid cycle MDHs are remarkably similar between eucarya and bacteria. MDH from Escherichia coli is 58% identical to porcine mitochondrial MDH w21x. Three-dimensional structural information is available for MDH from porcine heart mitochondria w22,23x, porcine heart cytoplasm w24x, the halophile Halobacterium marismortui w25x, E. coli w26x, and the thermophile Thermus flaÕus w27x. While dehydrogenases such as MDH which have evolved to function at elevated temperature or osmolarity have been examined in considerable detail, much less is known regarding the adaptation of en-
0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 6 . 0 0 2 0 0 - X
42
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
zyme function in general, and dehydrogenase function in particular, to decreased temperature or increased pressure. Partially purified MDH from the psychrophile Vibrio marinus lost activity when incubated above 208C or below 158C w28x. Protein sequence information from a psychrotrophic Bacillus species indicated that increased ion pair formation may be necessary for LDH function at low temperature w29x. Enzymes from psychrophilic organisms are typically characterized by higher specific activities and increased thermal sensitivities. These properties are attributed to a relative increase in flexibility w30x. While much remains to be learned about the molecular bases for enzyme adaptation to low temperature, a growing body of data is accumulating from the comparison of homologous proteins from mesophilic and psychrophilic sources w31–33x. Elevated pressure inhibits increases in system volume and thereby affects both the rates and equilibria of biochemical reactions in ways that are distinct from other physiochemical conditions. In many instances the volume changes which accompany these processes arise from hydration changes rather than alterations in the enzymes or substrates themselves w34x. Lactate dehydrogenases from deep-sea fishes display reduced catalytic efficiencies but greater inherent stabilities than homologous enzymes from related shallow water fishes w35,36x. Both MDHs and LDHs of deep-sea fishes and invertebrates have been found to possess Michaelis–Menten constants Žapparent K m . of cofactor and substrate which are much less perturbed by high pressure than are homologous enzymes of related shallow water organisms Žreviewed in w29x.. Amino-acid sequence comparisons of portions of LDHs isolated from congeneric fishes possessing high pressure adapted and high pressure sensitive properties indicate that the acquisition of pressure resistance must involve only minor changes in protein primary structure w37x. However, aminoacid substitutions which enhance enzyme function at high pressure have yet to be identified. In order to begin to clarify the molecular bases for dehydrogenase function at low temperature and at high pressure, we have cloned, sequenced and overexpressed the gene encoding malate dehydrogenase from the psychrophilic, barophilic deep-sea bacterium Photobacterium species strain SS9 w38x. Southern blot analysis w39x of SS9 chromosomal
Fig. 1. Overproduction of recombinant P. SS9 MDH. Lane 1, whole cell extract of E. coli DH5 a , lane 2, whole cell extract of the E. coli MDH deficient mutant W945TL-2, lane 3, E. coli W945TL-2 harboring plasmid pTW10. ŽA. Staining with Coomassie brilliant blue R to reveal the total protein profiles, ŽB. staining for MDH activity w46,47x. The 37 kDa recombinant MDH protein is indicated.
DNA resulted in the identification of a 2.5 kb EcoRI restriction fragment which strongly hybridized to a E. coli mdh gene probe w16x. Standard cloning protocols were used to clone this hybridizing DNA into M13mp18 w40x to generate pTW1. The 2.5 kb EcoRI fragment in pTW1 was subcloned into pUC18 w40x to generate pTW10. pTW10 was observed to confer high malate dehydrogenase activity to E. coli, including the E. coli malate dehydrogenase mutant W945TL-2 w41x. Production of recombinant MDH in E. coli was monitored by SDS-PAGE, protein renaturation, and malate dehydrogenase activity staining. Fig. 1A shows that pTW10 containing E. coli cells produced large amounts of a 37 kDa protein. Production of this protein occurred in the absence as well as presence of lac promoter induction, suggesting that expression was controlled by a promoter within the cloned DNA fragment. Malate dehydrogenase activity staining of proteins renatured following SDS-PAGE indicated that the 37 kDa protein possessed malate dehydrogenase enzyme activity ŽFig. 1B.. Finally, it was also possible to purify a 37 kDa protein with MDH activity from pTW10-containing E. coli cells or from Photobacterium species strain SS9 using the Matrix gel Red A affinity chromatography method of Smith
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
et al. w42x described for E. coli MDH purification Ždata not shown. . These results confirmed that the SS9 mdhA gene had been isolated. The position of the mdhA gene on the 2.5 kb EcoRI fragment was obtained following Sanger dideoxy DNA sequence analysis w43x and is shown in Fig. 2. Within a stretch of 1500 bp one open reading frame of 936 nucleotides encoding a protein of 32 kDa with homology to other MDHs was found. The discrepancy between the deduced and apparent molecular weight of the SS9 MDH is similar to that which has previously been reported for Vibrio 5710 w19x. Because the observed overproduction of recombinant SS9 MDH was similar to that reported for E.
Fig. 2. Nucleotide and deduced amino-acid sequences of the P. species strain SS9 mdhA gene. The underlined nucleotides highlight sequences shared with the upstream portion of the E. coli mdh gene or which indicate the ribosome binding sequence.
43
coli MDH when the E. coli mdh gene is present in multiple copies w17x, the upstream sequence of both genes were compared. Proximal to the SS9 mdhA start codon is found a ribosome binding sequence with a high degree of complementarity to E. coli 16S rRNA Žunderlined in Fig. 2.. Farther upstream, a 10 bp sequence beginning 168 bp upstream of the SS9 mdhA translational start Žalso underlined in Fig. 2. is identical to a sequence located between the E. coli mdh y35 and y10 promoter regions w17,44x. The E. coli mdh promoter sequence has been patented for its use in directing high levels of gene expression Ž patent number WO9308277-Ar2, 29 April, 1993.. In E. coli mdh gene expression is controlled by ArcA repression w45x, and catabolite repression w44x. However, no sequences bearing homology to the consensus binding sequences for ArcA or the cAMP receptor protein were identified in the SS9 mdhA upstream sequence. The SS9 MDH was compared to four other bacterial MDH sequences possessing similarity to the mitochondrial class w21x of MDHs Ž Fig. 3. . They represent three mesophilic sources, E. coli w16,17x, Salmonella typhimurium w15x, and Haemophilus influenzae w20x Žinferred from an open reading frame in its genome sequence. , and another MDH from a psychrophilic deep-sea isolate, Vibrio 5710 w19x. All of these MDHs are of identical size, 312 amino acids, except for the H. influenzae MDH which consists of 311 amino acids. In keeping with the close taxonomic relationship between the genera Photobacterium and Vibrio, Vibrio 5710 mdh shares the greatest identity with the SS9 mdhA coding sequence, 74% versus 68–72% for the other mdh sequences. However, at the protein level, Vibrio 5710 MDH shares the least identity with SS9 MDH, 76%, versus 79% for the enteric proteins. This is particularly curious because of the similar environments within which the Vibrio and Photobacterium MDHs must function. Among the 48 residues which are inferred from pig heart mitochondrial MDH and E. coli MDH crystallographic analyses to be involved in subunit binding, cofactor binding or catalysis w22,23,26x there are only 11 positions displaying heterogeneity among the 5 MDHs. The histidine at position 178 and the aspartic acid at position 150 are invariant active-site residues believed to serve as a proton relay during catalysis. Among the variable residues for which
44
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
function could be indicated there are two positions with SS9-specific substitutions, a leucine to isoleucine substitution at position 6 and a alanine to arginine substitution at position 219, both of which are involved in subunit binding. There is also a isoleucine for valine substitution at position 146 which is shared between the two pyschrophile MDHs and which is involved in nucleotide binding, and a serine for alanine substitution at position 159 which is unique to the psychrophile Vibrio 5710 and involved in subunit interaction. As previously described w19x, the MDHs
display greatest heterogeneity among the residues predicted to be exposed to the surface. In contrast to other psychrophilic proteins the deep-sea MDHs do not appear to possess a higher proportion of amino terminal stabilizing residues within their a-helices w33x nor do they display a smaller arginine to lysine ratio w32x. Detailed structurerfunction studies of the deep-sea MDHs will be required to determine residues important for protein stability or function at low temperature or high pressure.
Fig. 3. Alignment between Photobacterium species strain SS9 MDH and four other MDHs. The functions attributed to specific residues are based upon X-ray crystallographic information of porcine mitochondrial MDH and E. coli MDH w22,23,26x using the subscript designations previously utilized by Thompson et al. w12x. N, NADŽH. binding, Q, subunit interactions, C, catalysis. Deduced amino-acid sequences were aligned using the DNASYSTEM w48x programs SCORE, PREALIGN, and MULPUB. The following EMBLrGenBank accession numbers were used: Vibrio 5710, D78194; E. coli, Y00129; S. typhimurium, M95049; and H. influenzae, L42023.
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
This work was supported from an Academic Senate grant from the University of California, San Diego, and by grant N00014-90-J-1878 from the Office of Naval Research to D.H.B.
References w21x w1x Banaszak, L.J. and Bradshaw, R.A. Ž1975. Malate dehydrogenases, in The Enzymes ŽBoyer, P.D., ed.., Vol. XI, Oxidation–Reduction, Part A, pp. 369–396, Academic Press, New York. w2x Hecht, K. and Jaenicke, R. Ž1989. Biochemistry 28, 4979– 4985. w3x Hecht, K., Wrba, A. and Jaenicke, R. Ž1989. Eur. J. Biochem. 189, 63–74. w4x Ijima, S., Saiki, T. and Beppu, T. Ž1980. Biochim. Biophys. Acta 613, 1–9. w5x Sundaram, T.K., Wright, I.P. and Wilkinson, A.E. Ž1980. J. Am. Chem. Soc. 19, 2017– 2022. w6x Yoon, H. and Anderson, B.M. Ž1989. J. Gen. Microbiol. 135, 245–250. w7x Cretin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J.-C. and Pernollet, J.-C. Ž1990. Eur. J. Biochem. 192, 299–303. w8x Metzler, M.C., Rothermel, B.A. and Nelson, T. Ž1989. Plant Mol. Biol. 12, 713–722. w9x Setoyama, C., Joh, T., Tsuzuki, T. and Shimada, K. Ž1988. J. Mol. Biol. 202, 355–364. w10x Joh, T., Takeshima, H., Tsuzuki, T., Setoyama, C., Shimada, K., Tanase, S., Kuramitsu, S., Kagamiyama, H. and Morino, Y. Ž1987. J. Biol. Chem. 262, 15127–15131. w11x Joh, T., Takeshima, H., Tsuzuki, T., Shimada, K., Tanase, S. and Morino, Y. Ž1987. Biochemistry 26, 2515–2520. w12x Thompson, L.M., Sutherland, P., Steffan, J.S. and McAllister-Henn, L. Ž1988. Biochemistry 27, 8393–8400. w13x Gorisch, H. and Jany, K.-D. Ž1989. FEBS Lett. 247, 259– 262. w14x Honka, E., Fabry, S., Niermann, T., Palm, P. and Hensel, R. Ž1989. Eur. J. Biochem. 188, 623–632. w15x Lu, C.-D. and Abdelal, A.T. Ž1993. Gene 123, 143–144. w16x McAllister-Henn, L., Blaber, M., Bradshaw, R.A. and Nisco, S.J. Ž1987. Nucleic Acids Res. 15, 4993. w17x Nicholls, D.J., Minton, N.P., Atkinson, T. and Sundaram, T.K. Ž1989. Appl. Microb. Biotechnol. 31, 376–382. w18x Nishiyama, M., Matsubara, N., Yamamoto, K., Iijima, S., Uozumi, T. and Beppu, T. Ž1986. J. Biol. Chem. 261, 14178–14183. w19x Okhuma, M., Ohtoko, K., Takada, N., Hamamoto, T., Usami, R., Kudo, T. and Horikoshi, K. Ž1996. FEMS Microbiol. Lett. 137, 247–252. w20x Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.-F.,
w22x w23x w24x w25x
w26x w27x w28x w29x w30x
w31x w32x w33x
w34x w35x w36x w37x w38x w39x w40x w41x w42x w43x
45
Dougherty, B.A., Merrick, J.M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C., Gocayne, J.D., Scott, J., Shirley, R., Liu, L.-I., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips, C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Hanna, M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L.D., Fritchman, J.L., Fuhrmann, J.L., Geoghagen, N.S.M., Gnehm, C.L., McDonald, L.A., Small, K.V., Fraser, C.M., Smith, H.O. and Venter, J.C. Ž1995. Science 269, 496–512. McAlister-Henn, L. Ž1988. Trends Biochem. Sci. 13, 178– 181. Roderick, S.L. and Banaszak, L.L. Ž1986. J. Biol. Chem. 261, 9461–9464. Birktoft, J.J., Bradshaw, R.A. and Banaszak, L.J. Ž1987. Biochemistry 26, 2722–2734. Birktoft, J.J., Rhodes, G. and Banaszak, L.J. Ž1989. Biochemistry 28, 6065–6081. Harel, M., Shoham, M., Frolow, F., Eisenberg, H., Mevarech, M., Yonath, A. and Sussman, J.L. Ž1988. J. Mol. Biol. 200, 609–610. Hall, M.D., Levitt, D.G. and Banaszak L.J. Ž1992. J. Mol. Biol. 226, 867–882. Kelly, C.A., Sarfaty, S., Nishiyama, M., Beppu, T. and Birktoft, J.J. Ž1991. J. Mol. Biol. 221, 383–385. Langridge, P. and Morita, R.Y. Ž1966. J. Bacteriol. 92, 418–423. Zuber, H. Ž1988. Biophys. Chem. 29, 171–179. Somero, G.N. Ž1991. in Environmental and Metabolic Animal Physiology ŽProsser, C.L., ed.., pp. 167–204, WileyLiss, New York. Schlatter, D., Kriech, O., Suter, F. and Zuber, H. Ž1987. Biol. Chem. Hoppe-Seyler 368, 1435–1446. Arpigny, J.L., Feller, G. and Gerday, C. Ž1993. Biochim. Biophys. Acta 1171, 331–333. Rentiere-Delrue, F., Mande, S.C., Moyens, S., Terpstra, P., Mainfroid, V., Goraj, K., Lion, M., Hol, W.G.J. and Martial, J.A. Ž1993. J. Mol. Biol. 229, 85–93. Somero, G.N. Ž1992. Annu. Rev. Physiol. 54, 557–577. Yancey, P.H. and Siebenaller, J.F. Ž1987. Biochim. Biophys. Acta 924, 483–491. Hennessey, J.P. and Siebenaller, J.F. Ž1987. J. Exp. Zool. 241, 9–15. Siebenaller, J.F. Ž1984. Biochim. Biophys. Acta 786, 161– 169. Bartlett, D., Wright, M., Yayaynos, A.A. and Silverman, M. Ž1989. Nature 342, 572–574. Southern, E.M. Ž1975. J. Mol. Biol. 98, 503–517. Norrander, J., Kempe, T. and Messing, J. Ž1983. Gene 26, 101–106. Courtright, J.B. and Henning, U. Ž1970. J. Bacteriol. 102, 722–728. Smith, K., Sundaram, T.K., Kernick, M. and Wilkinson, A.E. Ž1982. Biochim. Biophys. Acta 708, 17–25. Sanger, F., Nicklen, S. and Coulson, A.R. Ž1977. Proc. Natl. Acad. Sci. USA 74, 5463–5467.
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w44x Vogel, R.F., Entian, K.-D. and Mecke, D. Ž1987. Arch. Microbiol. 149, 36–42. w45x Park, S.-J., Cotter, P.A. and Gunsalus, R.P. Ž1995. J. Bacteriol. 177, 6652–6656. w46x Lacks, S.A. and Springhorn, S.S. Ž1980. J. Biol. Chem. 255, 7467–7473.
w47x Dietz, A. and Lubrano, T. Ž1967. Anal. Biochem. 20, 246– 257. w48x Smith, D.W. Ž1988. Computer Appl. Biosci. 4, 212–220.
Short sequence-paper
Cloning, sequencing and overexpression of the gene encoding malate dehydrogenase from the deep-sea bacterium Photobacterium species strain SS9 1 Timothy J. Welch, Douglas H. Bartlett
)
Center for Marine Biotechnology and Biomedicine, Marine Biology Research DiÕision, Scripps Institution of Oceanography, UniÕersity of California, 9500 Gilman DriÕe, San Diego, La Jolla, CA 92093-0202, USA Received 25 July 1996; revised 10 October 1996; accepted 14 October 1996
Abstract The gene encoding malate dehydrogenase Ž mdhA. was obtained from the psychrophilic, barophilic, deep-sea isolate Photobacterium species strain SS9. The SS9 mdhA gene directed high levels of malate dehydrogenase ŽMDH. production in Escherichia coli. A comparison of SS9 MDH to three mesophile MDHs, a MDH sequence obtained from another deep-sea bacterium, and to other psychrophile proteins is presented. Keywords: Psychrophile; Barophile; Cold adaptation; High pressure adaptation; TCA cycle; Malate dehydrogenase; Ž Photobacterium.; Ž E. coli .
Malate dehydrogenase ŽMDH; L-malate-nicotine adenine dinucleotide wNADx oxidoreductase, EC 1.1.1.37., catalyzes the NADŽH. -dependent interconversion of malate and oxaloacetate. Although MDHs exist in different isozyme forms, and possess roles in diverse metabolic activities including aspartate biosynthesis, the malate-aspartate shuttle, gluconeogenesis, and lipogenesis, MDH is best known as an enzyme of the tricarboxylic acid cycle w1x. Along with lactate dehydrogenase Ž LDH. , another 2-hydroxy acid dehydrogenase, as well as other dehydrogenases, MDH has been one of the premier enzymes used for examining protein biochemical adaptation to
)
Corresponding author. Fax: q1 Ž619. 5347313; E-mail: [email protected] 1 The sequence data reported in this paper is present in the GenBankr EMBL libraries under the accession number L13319.
extreme environments. For example, kinetic information exists for MDHs from a variety of mesophilic, thermophilic, and halophilic microorganisms Žexamples in Refs. w2–6x.. Numerous malate dehydrogenase gene sequences and protein sequences are also available from Eucarya w7–12x, Archaea w13,14x, and Bacteria w17–20x. The amino-acid sequences of tricarboxylic acid cycle MDHs are remarkably similar between eucarya and bacteria. MDH from Escherichia coli is 58% identical to porcine mitochondrial MDH w21x. Three-dimensional structural information is available for MDH from porcine heart mitochondria w22,23x, porcine heart cytoplasm w24x, the halophile Halobacterium marismortui w25x, E. coli w26x, and the thermophile Thermus flaÕus w27x. While dehydrogenases such as MDH which have evolved to function at elevated temperature or osmolarity have been examined in considerable detail, much less is known regarding the adaptation of en-
0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 6 . 0 0 2 0 0 - X
42
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
zyme function in general, and dehydrogenase function in particular, to decreased temperature or increased pressure. Partially purified MDH from the psychrophile Vibrio marinus lost activity when incubated above 208C or below 158C w28x. Protein sequence information from a psychrotrophic Bacillus species indicated that increased ion pair formation may be necessary for LDH function at low temperature w29x. Enzymes from psychrophilic organisms are typically characterized by higher specific activities and increased thermal sensitivities. These properties are attributed to a relative increase in flexibility w30x. While much remains to be learned about the molecular bases for enzyme adaptation to low temperature, a growing body of data is accumulating from the comparison of homologous proteins from mesophilic and psychrophilic sources w31–33x. Elevated pressure inhibits increases in system volume and thereby affects both the rates and equilibria of biochemical reactions in ways that are distinct from other physiochemical conditions. In many instances the volume changes which accompany these processes arise from hydration changes rather than alterations in the enzymes or substrates themselves w34x. Lactate dehydrogenases from deep-sea fishes display reduced catalytic efficiencies but greater inherent stabilities than homologous enzymes from related shallow water fishes w35,36x. Both MDHs and LDHs of deep-sea fishes and invertebrates have been found to possess Michaelis–Menten constants Žapparent K m . of cofactor and substrate which are much less perturbed by high pressure than are homologous enzymes of related shallow water organisms Žreviewed in w29x.. Amino-acid sequence comparisons of portions of LDHs isolated from congeneric fishes possessing high pressure adapted and high pressure sensitive properties indicate that the acquisition of pressure resistance must involve only minor changes in protein primary structure w37x. However, aminoacid substitutions which enhance enzyme function at high pressure have yet to be identified. In order to begin to clarify the molecular bases for dehydrogenase function at low temperature and at high pressure, we have cloned, sequenced and overexpressed the gene encoding malate dehydrogenase from the psychrophilic, barophilic deep-sea bacterium Photobacterium species strain SS9 w38x. Southern blot analysis w39x of SS9 chromosomal
Fig. 1. Overproduction of recombinant P. SS9 MDH. Lane 1, whole cell extract of E. coli DH5 a , lane 2, whole cell extract of the E. coli MDH deficient mutant W945TL-2, lane 3, E. coli W945TL-2 harboring plasmid pTW10. ŽA. Staining with Coomassie brilliant blue R to reveal the total protein profiles, ŽB. staining for MDH activity w46,47x. The 37 kDa recombinant MDH protein is indicated.
DNA resulted in the identification of a 2.5 kb EcoRI restriction fragment which strongly hybridized to a E. coli mdh gene probe w16x. Standard cloning protocols were used to clone this hybridizing DNA into M13mp18 w40x to generate pTW1. The 2.5 kb EcoRI fragment in pTW1 was subcloned into pUC18 w40x to generate pTW10. pTW10 was observed to confer high malate dehydrogenase activity to E. coli, including the E. coli malate dehydrogenase mutant W945TL-2 w41x. Production of recombinant MDH in E. coli was monitored by SDS-PAGE, protein renaturation, and malate dehydrogenase activity staining. Fig. 1A shows that pTW10 containing E. coli cells produced large amounts of a 37 kDa protein. Production of this protein occurred in the absence as well as presence of lac promoter induction, suggesting that expression was controlled by a promoter within the cloned DNA fragment. Malate dehydrogenase activity staining of proteins renatured following SDS-PAGE indicated that the 37 kDa protein possessed malate dehydrogenase enzyme activity ŽFig. 1B.. Finally, it was also possible to purify a 37 kDa protein with MDH activity from pTW10-containing E. coli cells or from Photobacterium species strain SS9 using the Matrix gel Red A affinity chromatography method of Smith
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
et al. w42x described for E. coli MDH purification Ždata not shown. . These results confirmed that the SS9 mdhA gene had been isolated. The position of the mdhA gene on the 2.5 kb EcoRI fragment was obtained following Sanger dideoxy DNA sequence analysis w43x and is shown in Fig. 2. Within a stretch of 1500 bp one open reading frame of 936 nucleotides encoding a protein of 32 kDa with homology to other MDHs was found. The discrepancy between the deduced and apparent molecular weight of the SS9 MDH is similar to that which has previously been reported for Vibrio 5710 w19x. Because the observed overproduction of recombinant SS9 MDH was similar to that reported for E.
Fig. 2. Nucleotide and deduced amino-acid sequences of the P. species strain SS9 mdhA gene. The underlined nucleotides highlight sequences shared with the upstream portion of the E. coli mdh gene or which indicate the ribosome binding sequence.
43
coli MDH when the E. coli mdh gene is present in multiple copies w17x, the upstream sequence of both genes were compared. Proximal to the SS9 mdhA start codon is found a ribosome binding sequence with a high degree of complementarity to E. coli 16S rRNA Žunderlined in Fig. 2.. Farther upstream, a 10 bp sequence beginning 168 bp upstream of the SS9 mdhA translational start Žalso underlined in Fig. 2. is identical to a sequence located between the E. coli mdh y35 and y10 promoter regions w17,44x. The E. coli mdh promoter sequence has been patented for its use in directing high levels of gene expression Ž patent number WO9308277-Ar2, 29 April, 1993.. In E. coli mdh gene expression is controlled by ArcA repression w45x, and catabolite repression w44x. However, no sequences bearing homology to the consensus binding sequences for ArcA or the cAMP receptor protein were identified in the SS9 mdhA upstream sequence. The SS9 MDH was compared to four other bacterial MDH sequences possessing similarity to the mitochondrial class w21x of MDHs Ž Fig. 3. . They represent three mesophilic sources, E. coli w16,17x, Salmonella typhimurium w15x, and Haemophilus influenzae w20x Žinferred from an open reading frame in its genome sequence. , and another MDH from a psychrophilic deep-sea isolate, Vibrio 5710 w19x. All of these MDHs are of identical size, 312 amino acids, except for the H. influenzae MDH which consists of 311 amino acids. In keeping with the close taxonomic relationship between the genera Photobacterium and Vibrio, Vibrio 5710 mdh shares the greatest identity with the SS9 mdhA coding sequence, 74% versus 68–72% for the other mdh sequences. However, at the protein level, Vibrio 5710 MDH shares the least identity with SS9 MDH, 76%, versus 79% for the enteric proteins. This is particularly curious because of the similar environments within which the Vibrio and Photobacterium MDHs must function. Among the 48 residues which are inferred from pig heart mitochondrial MDH and E. coli MDH crystallographic analyses to be involved in subunit binding, cofactor binding or catalysis w22,23,26x there are only 11 positions displaying heterogeneity among the 5 MDHs. The histidine at position 178 and the aspartic acid at position 150 are invariant active-site residues believed to serve as a proton relay during catalysis. Among the variable residues for which
44
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
function could be indicated there are two positions with SS9-specific substitutions, a leucine to isoleucine substitution at position 6 and a alanine to arginine substitution at position 219, both of which are involved in subunit binding. There is also a isoleucine for valine substitution at position 146 which is shared between the two pyschrophile MDHs and which is involved in nucleotide binding, and a serine for alanine substitution at position 159 which is unique to the psychrophile Vibrio 5710 and involved in subunit interaction. As previously described w19x, the MDHs
display greatest heterogeneity among the residues predicted to be exposed to the surface. In contrast to other psychrophilic proteins the deep-sea MDHs do not appear to possess a higher proportion of amino terminal stabilizing residues within their a-helices w33x nor do they display a smaller arginine to lysine ratio w32x. Detailed structurerfunction studies of the deep-sea MDHs will be required to determine residues important for protein stability or function at low temperature or high pressure.
Fig. 3. Alignment between Photobacterium species strain SS9 MDH and four other MDHs. The functions attributed to specific residues are based upon X-ray crystallographic information of porcine mitochondrial MDH and E. coli MDH w22,23,26x using the subscript designations previously utilized by Thompson et al. w12x. N, NADŽH. binding, Q, subunit interactions, C, catalysis. Deduced amino-acid sequences were aligned using the DNASYSTEM w48x programs SCORE, PREALIGN, and MULPUB. The following EMBLrGenBank accession numbers were used: Vibrio 5710, D78194; E. coli, Y00129; S. typhimurium, M95049; and H. influenzae, L42023.
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
This work was supported from an Academic Senate grant from the University of California, San Diego, and by grant N00014-90-J-1878 from the Office of Naval Research to D.H.B.
References w21x w1x Banaszak, L.J. and Bradshaw, R.A. Ž1975. Malate dehydrogenases, in The Enzymes ŽBoyer, P.D., ed.., Vol. XI, Oxidation–Reduction, Part A, pp. 369–396, Academic Press, New York. w2x Hecht, K. and Jaenicke, R. Ž1989. Biochemistry 28, 4979– 4985. w3x Hecht, K., Wrba, A. and Jaenicke, R. Ž1989. Eur. J. Biochem. 189, 63–74. w4x Ijima, S., Saiki, T. and Beppu, T. Ž1980. Biochim. Biophys. Acta 613, 1–9. w5x Sundaram, T.K., Wright, I.P. and Wilkinson, A.E. Ž1980. J. Am. Chem. Soc. 19, 2017– 2022. w6x Yoon, H. and Anderson, B.M. Ž1989. J. Gen. Microbiol. 135, 245–250. w7x Cretin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J.-C. and Pernollet, J.-C. Ž1990. Eur. J. Biochem. 192, 299–303. w8x Metzler, M.C., Rothermel, B.A. and Nelson, T. Ž1989. Plant Mol. Biol. 12, 713–722. w9x Setoyama, C., Joh, T., Tsuzuki, T. and Shimada, K. Ž1988. J. Mol. Biol. 202, 355–364. w10x Joh, T., Takeshima, H., Tsuzuki, T., Setoyama, C., Shimada, K., Tanase, S., Kuramitsu, S., Kagamiyama, H. and Morino, Y. Ž1987. J. Biol. Chem. 262, 15127–15131. w11x Joh, T., Takeshima, H., Tsuzuki, T., Shimada, K., Tanase, S. and Morino, Y. Ž1987. Biochemistry 26, 2515–2520. w12x Thompson, L.M., Sutherland, P., Steffan, J.S. and McAllister-Henn, L. Ž1988. Biochemistry 27, 8393–8400. w13x Gorisch, H. and Jany, K.-D. Ž1989. FEBS Lett. 247, 259– 262. w14x Honka, E., Fabry, S., Niermann, T., Palm, P. and Hensel, R. Ž1989. Eur. J. Biochem. 188, 623–632. w15x Lu, C.-D. and Abdelal, A.T. Ž1993. Gene 123, 143–144. w16x McAllister-Henn, L., Blaber, M., Bradshaw, R.A. and Nisco, S.J. Ž1987. Nucleic Acids Res. 15, 4993. w17x Nicholls, D.J., Minton, N.P., Atkinson, T. and Sundaram, T.K. Ž1989. Appl. Microb. Biotechnol. 31, 376–382. w18x Nishiyama, M., Matsubara, N., Yamamoto, K., Iijima, S., Uozumi, T. and Beppu, T. Ž1986. J. Biol. Chem. 261, 14178–14183. w19x Okhuma, M., Ohtoko, K., Takada, N., Hamamoto, T., Usami, R., Kudo, T. and Horikoshi, K. Ž1996. FEMS Microbiol. Lett. 137, 247–252. w20x Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.-F.,
w22x w23x w24x w25x
w26x w27x w28x w29x w30x
w31x w32x w33x
w34x w35x w36x w37x w38x w39x w40x w41x w42x w43x
45
Dougherty, B.A., Merrick, J.M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C., Gocayne, J.D., Scott, J., Shirley, R., Liu, L.-I., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips, C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Hanna, M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L.D., Fritchman, J.L., Fuhrmann, J.L., Geoghagen, N.S.M., Gnehm, C.L., McDonald, L.A., Small, K.V., Fraser, C.M., Smith, H.O. and Venter, J.C. Ž1995. Science 269, 496–512. McAlister-Henn, L. Ž1988. Trends Biochem. Sci. 13, 178– 181. Roderick, S.L. and Banaszak, L.L. Ž1986. J. Biol. Chem. 261, 9461–9464. Birktoft, J.J., Bradshaw, R.A. and Banaszak, L.J. Ž1987. Biochemistry 26, 2722–2734. Birktoft, J.J., Rhodes, G. and Banaszak, L.J. Ž1989. Biochemistry 28, 6065–6081. Harel, M., Shoham, M., Frolow, F., Eisenberg, H., Mevarech, M., Yonath, A. and Sussman, J.L. Ž1988. J. Mol. Biol. 200, 609–610. Hall, M.D., Levitt, D.G. and Banaszak L.J. Ž1992. J. Mol. Biol. 226, 867–882. Kelly, C.A., Sarfaty, S., Nishiyama, M., Beppu, T. and Birktoft, J.J. Ž1991. J. Mol. Biol. 221, 383–385. Langridge, P. and Morita, R.Y. Ž1966. J. Bacteriol. 92, 418–423. Zuber, H. Ž1988. Biophys. Chem. 29, 171–179. Somero, G.N. Ž1991. in Environmental and Metabolic Animal Physiology ŽProsser, C.L., ed.., pp. 167–204, WileyLiss, New York. Schlatter, D., Kriech, O., Suter, F. and Zuber, H. Ž1987. Biol. Chem. Hoppe-Seyler 368, 1435–1446. Arpigny, J.L., Feller, G. and Gerday, C. Ž1993. Biochim. Biophys. Acta 1171, 331–333. Rentiere-Delrue, F., Mande, S.C., Moyens, S., Terpstra, P., Mainfroid, V., Goraj, K., Lion, M., Hol, W.G.J. and Martial, J.A. Ž1993. J. Mol. Biol. 229, 85–93. Somero, G.N. Ž1992. Annu. Rev. Physiol. 54, 557–577. Yancey, P.H. and Siebenaller, J.F. Ž1987. Biochim. Biophys. Acta 924, 483–491. Hennessey, J.P. and Siebenaller, J.F. Ž1987. J. Exp. Zool. 241, 9–15. Siebenaller, J.F. Ž1984. Biochim. Biophys. Acta 786, 161– 169. Bartlett, D., Wright, M., Yayaynos, A.A. and Silverman, M. Ž1989. Nature 342, 572–574. Southern, E.M. Ž1975. J. Mol. Biol. 98, 503–517. Norrander, J., Kempe, T. and Messing, J. Ž1983. Gene 26, 101–106. Courtright, J.B. and Henning, U. Ž1970. J. Bacteriol. 102, 722–728. Smith, K., Sundaram, T.K., Kernick, M. and Wilkinson, A.E. Ž1982. Biochim. Biophys. Acta 708, 17–25. Sanger, F., Nicklen, S. and Coulson, A.R. Ž1977. Proc. Natl. Acad. Sci. USA 74, 5463–5467.
46
T.J. Welch, D.H. Bartlettr Biochimica et Biophysica Acta 1350 (1997) 41–46
w44x Vogel, R.F., Entian, K.-D. and Mecke, D. Ž1987. Arch. Microbiol. 149, 36–42. w45x Park, S.-J., Cotter, P.A. and Gunsalus, R.P. Ž1995. J. Bacteriol. 177, 6652–6656. w46x Lacks, S.A. and Springhorn, S.S. Ž1980. J. Biol. Chem. 255, 7467–7473.
w47x Dietz, A. and Lubrano, T. Ž1967. Anal. Biochem. 20, 246– 257. w48x Smith, D.W. Ž1988. Computer Appl. Biosci. 4, 212–220.