Skip to main content

Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase

Molecular Genetics and Genomics volume 275, pages 74–80 (2006)Cite this article

Abstract

Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + α-ketoglutarate) of the novel α-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-α-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (Canada)

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Ausubel FM et al (1998) Current protocols in molecular biology. Wiley, New York

    Google Scholar 

  • Bhattacharjee JK (1992) Evolution of α-aminoadipate pathway for the synthesis of lysine in fungi. In: Mortlock RP (ed) Evolution of metabolic function. CRC Press, Boca Raton, pp 47–80

    Google Scholar 

  • Bhattacharjee JK, Strassmen M (1967) Accumulation of tricarboxylic acids related to lysine biosynthesis in a yeast mutant. J Biol Chem 242:2542–2546

    PubMed  CAS  Google Scholar 

  • Bordo D, Argos P (1991) Suggestions for “safe” residue substitutions in site-directed mutagenesis. J Mol Biol 217:721–729

    Article  PubMed  CAS  Google Scholar 

  • Broquist HP (1971) Lysine biosynthesis in yeast. Methods Enzymol 17B:112–129

    Article  Google Scholar 

  • Busch S, Bode HB, Brakhage AA, Braus GH (2003) Impact of the cross-pathway control on the regulation of lysine and penicillin biosynthesis in Aspergillus nidulans. Curr Genet 42:209–219

    PubMed  CAS  Google Scholar 

  • Churcher C, Bowman S, Badcock K et al (1997) The nucleotide sequence of Saccharomyces cerevisiae chromosome IX. Nature 387(6632 Suppl):84–87

    PubMed  CAS  Google Scholar 

  • Dancis J, Hutzler J, Woody NC, Cox RP (1976) Multiple enzyme defects in familial hyperlysinemia. Pediatr Res 10:686–691

    Article  PubMed  CAS  Google Scholar 

  • Feller A, Dubois E, Ramos F, Pierard A (1994) Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation. Mol Cell Biol 14:6411–6418

    PubMed  CAS  Google Scholar 

  • Fujioka M, Takata Y, Ogawa H, Okamoto M (1980) The inactivation of saccharopine dehydrogenase (l-lysine-forming) by diethyl pyrocarbonate. J Biol Chem 255:937–942

    PubMed  CAS  Google Scholar 

  • Garrad RC, Bhattacharjee JK (1992) Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans. J Bacteriol 174:7379–7384

    PubMed  CAS  Google Scholar 

  • Garrad RC, Schmidt TM, Bhattacharjee JK (1994) Molecular and functional analysis of the LYS1 gene of Candida albicans. Infect Immun 62:5027–5031

    PubMed  CAS  Google Scholar 

  • Gaziola SA, Teixeira CM, Lugli J, Sodek L, Azevedo RA (1997) The enzymology of lysine catabolism in rice seeds—isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase/saccharopine dehydrogenase bifunctional polypeptide. Eur J Biochem 247:364–371

    Article  PubMed  CAS  Google Scholar 

  • Guo S, Bhattacharjee JK (2003) Site-directed mutational analysis of the novel catalytic domains of α-aminoadipate reductase (Lys2p) from Candida albicans. Mol Genet Genomics (MGG) 269:271–279

    CAS  Google Scholar 

  • Hwang YL, Lindegren G, Lindegren CC (1966) Genetic study of lysine biosynthesis in yeast. Can J Genet Cytol 8:471–480

    CAS  Google Scholar 

  • Irvin SD, Bhattacharjee JK (1998) A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms. J Mol Evol 46:401–408

    Article  PubMed  CAS  Google Scholar 

  • Johansson E, Steffens JJ, Lindqvist Y, Schneider G (2000) Crystal structure of saccharopine reductase from Magnaporthe grisea, an enzyme of the alpha-aminoadipate pathway of lysine biosynthesis. Structure Fold Des 8:1037–1047

    Article  PubMed  CAS  Google Scholar 

  • Jones EE, Broquist HP (1965) Saccharopine, an intermediate of the aminoadipic acid pathway of lysine biosynthesis. II. Studies in Saccharomyces cerviseae. J Biol Chem 240:2531–2536

    PubMed  CAS  Google Scholar 

  • Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acid Res 30:3059–3066

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: Molecular evolutionary genetics analysis software. Arizona State University, Tempe

    Google Scholar 

  • Markovitz PJ, Chuang DT, Cox RP (1984) Familial hyperlysinemias. Purification and characterization of the bifunctional aminoadipic semialdehyde synthase with lysine-ketoglutarate reductase and saccharopine dehydrogenase activities. J Biol Chem 259:11643–11646

    PubMed  CAS  Google Scholar 

  • Naranjo L, Martin de Valmaseda E, Banuelos O, Lopez P, Riano J, Casqueiro J, Martin JF (2001) Conversion of pipecolic acid into lysine in Penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase. J Bacteriol 183:7165–7172

    Article  PubMed  CAS  Google Scholar 

  • Natarajan K, Meyer MR, Jackson BM, Slade D, Roberts C, Hinnebusch AG, Marton MJ (2001) Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol Cell Biol 21:4347–4368

    Article  PubMed  CAS  Google Scholar 

  • Nishida H, Nishiyama M (2000) What is characteristic of fungal lysine synthesis through the alpha-aminoadipate pathway? J Mol Evol 51:299–302

    PubMed  CAS  Google Scholar 

  • Ogawa H, Fujioka M (1978) Purification and characterization of saccharopine dehydrogenase from baker’s yeast. J Biol Chem 253:3666–3670

    PubMed  CAS  Google Scholar 

  • Ogawa H, Hase T, Fujioka M (1980) Amino acid sequence of a peptide containing an essential cysteine residue of yeast saccharopine dehydrogenase (l-lysine-forming). Biochim Biophys Acta 623:225–228

    PubMed  CAS  Google Scholar 

  • Papes F, Kemper EL, Cord-Neto G, Langone F, Arruda P (1999) Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse. Biochem J 344:555–563

    Article  PubMed  CAS  Google Scholar 

  • Sacksteder KA, Biery BJ, Morrell JC, Goodman BK, Geisbrecht BV, Cox RP, Gould SJ, Geraghty MT (2000) Identification of the alpha-aminoadipic semialdehyde synthase gene, which is defective in familial hyperlysinemia. Am J Hum Genet 66:1736–1743

    Article  PubMed  CAS  Google Scholar 

  • Urrestarazu LA, Borell CW, Bhattacharjee JK (1985) General and specific controls of lysine biosynthesis in Saccharomyces cerevisiae. Curr Genet 9:341–344

    Article  PubMed  CAS  Google Scholar 

  • van Burik JA, Magee PT (2001) Aspects of fungal pathogenesis in humans. Annu Rev Microbiol 55:743–772

    Article  PubMed  Google Scholar 

  • Vogel HJ (1960) Two modes of lysine synthesis among lower fungal. Biochem Biophys Acta 41:172–174

    Article  CAS  Google Scholar 

  • Winston MK, Bhattacharjee JK (1982) Growth inhibition by alpha-aminoadipate and reversal of the effect by specific amino acid supplements in Saccharomyces cerevisiae. J Bacteriol 152:874–879

    PubMed  CAS  Google Scholar 

  • Wood V, Gwilliam R, Rajandream MA et al (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415:871–880

    Article  PubMed  CAS  Google Scholar 

  • Xuan JW, Fournier P, Declerck N, Chasles M, Gaillardin C (1990) Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica. Mol Cell Biol 10:4795–4806

    PubMed  CAS  Google Scholar 

  • Ye ZH, Garrad RC, Winston MK, Bhattacharjee JK (1991) Use of alpha-aminoadipate and lysine as sole nitrogen source by Schizosaccharomyces pombe and selected pathogenic fungi. J Basic Microbiol 31:149–156

    Article  PubMed  CAS  Google Scholar 

  • Zabriskie TM, Jackson MD (2000) Lysine biosynthesis and metabolism in fungi. Nat Prod Rep17:85–97

    Article  Google Scholar 

  • Zhu X, Tang G, Galili G (2002) The activity of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is regulated by functional interaction between its two enzyme domains. J Biol Chem 277:49655–49661

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank G.R. Janssen for his valuable comments on the manuscript, X.F. Wan for computer support and Barbara J. Stahl for the preparation of the manuscript. This research was supported by a National Institute of General Medical Science grant 1R15GM065153 and Miami University to J.K.B. and a National Institute of General Medical Science grant 1R15GM068547 and Missouri State University to R.C.G.

Author information

Authors and Affiliations

  1. Department of Microbiology, Miami University, Pearson Hall, Room 46, Oxford, OH, 45056, USA

    Shujuan Guo & J. K. Bhattacharjee

  2. Biomedical Sciences Department, Missouri State University, 901 South National, Springfield, MO, 65804, USA

    Richard C. Garrad

Authors
  1. Shujuan Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard C. Garrad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. K. Bhattacharjee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. K. Bhattacharjee.

Additional information

Communicated by F. Messenguy

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guo, S., Garrad, R.C. & Bhattacharjee, J.K. Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase. Mol Genet Genomics 275, 74–80 (2006). https://doi.org/10.1007/s00438-005-0062-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-005-0062-z

Keywords

  • C . albicans
  • Saccharopine dehydrogenase
  • Lys1p SDH
  • Site-directed mutation
  • Phylogeny