Cold-Adapted Alanine Dehydrogenases from Two Antarctic Bacterial Strains: Gene Cloning, Protein Characterization, and Comparison with Mesophilic and Thermophilic Counterparts

ABSTRACT The genes encoding NAD+-dependent alanine dehydrogenases (AlaDHs) (EC 1.4.1.1 ) from the Antarctic bacterial organisms Shewanella sp. strain Ac10 (SheAlaDH) andCarnobacterium sp. strain St2 (CarAlaDH) were cloned and expressed in Escherichia coli. Of all of the AlaDHs that have been sequenced, SheAlaDH exhibited the highest level of sequence similarity to the AlaDH from the gram-negative bacterium Vibrio proteolyticus (VprAlaDH). CarAlaDH was most similar to AlaDHs from mesophilic and thermophilic Bacillus strains. SheAlaDH and CarAlaDH had features typical of cold-adapted enzymes; both the optimal temperature for catalytic activity and the temperature limit for retaining thermostability were lower than the values obtained for the mesophilic counterparts. Thekcat/Km value for the SheAlaDH reaction was about three times higher than thekcat/Km value for VprAlaDH, but it was much lower than thekcat/Km value for the AlaDH from Bacillus subtilis. Homology-based structural models of various AlaDHs, including the two psychrotrophic AlaDHs, were constructed. The thermal instability of SheAlaDH and CarAlaDH may result from relatively low numbers of salt bridges in these proteins.

[1]  K. Soda,et al.  Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus. , 1999, Biochemical and biophysical research communications.

[2]  A. Grossman,et al.  Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis , 1993, Journal of bacteriology.

[3]  J. Lamotte‐Brasseur,et al.  Cold adaptation parameters derived from cDNA sequencing and molecular modelling of elastase from Antarctic fish Notothenia neglecta. , 1997, Protein engineering.

[4]  Lode Wyns,et al.  Triose-phosphate Isomerase (TIM) of the Psychrophilic BacteriumVibrio marinus , 1998, The Journal of Biological Chemistry.

[5]  P Argos,et al.  Thermal stability and protein structure. , 1979, Biochemistry.

[6]  C. Wu,et al.  Back-propagation and counter-propagation neural networks for phylogenetic classification of ribosomal RNA sequences. , 1994, Nucleic acids research.

[7]  G. Feller,et al.  Psychrophilic enzymes: a thermodynamic challenge. , 1997, Biochimica et biophysica acta.

[8]  C. Arnold,et al.  MEGALIGN. The multiple alignment module of LASERGENE. , 1997, Methods in molecular biology.

[9]  J. Thornton,et al.  Ion-pairs in proteins. , 1983, Journal of molecular biology.

[10]  M. Kula,et al.  Dehydrogenases for the synthesis of chiral compounds. , 1989, European journal of biochemistry.

[11]  P. Andersen,et al.  Structure and function of a 40,000-molecular-weight protein antigen of Mycobacterium tuberculosis , 1992, Infection and immunity.

[12]  L. Wyns,et al.  Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties. , 1998, The Journal of biological chemistry.

[13]  B L Maidak,et al.  The RDP-II (Ribosomal Database Project) , 2001, Nucleic Acids Res..

[14]  A. Szilágyi,et al.  Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling. , 1995, Protein engineering.

[15]  G A Petsko,et al.  Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp. I5 suggest reasons for thermal instability. , 1997, Protein engineering.

[16]  N. Willassen,et al.  Cold adapted enzymes. , 2000, Biotechnology annual review.

[17]  P Argos,et al.  Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. , 1989, Journal of molecular biology.

[18]  K. Soda,et al.  Thermolabile alanine racemase from a psychotroph, Pseudomonas fluorescens: purification and properties. , 1993, Bioscience, biotechnology, and biochemistry.

[19]  G A Petsko,et al.  Aromatic-aromatic interaction: a mechanism of protein structure stabilization. , 1985, Science.

[20]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[21]  Ross A. Overbeek,et al.  The RDP (Ribosomal Database Project) , 1997, Nucleic Acids Res..

[22]  K. Soda,et al.  Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes , 1997, Applied and environmental microbiology.

[23]  R. Haser,et al.  Structures of the psychrophilic Alteromonas haloplanctis alpha-amylase give insights into cold adaptation at a molecular level. , 1998, Structure.

[24]  R. Y. Morita,et al.  PSYCHROPHILIC BACTERIA , 1959, Bacteriological reviews.

[25]  A Ikai,et al.  Thermostability and aliphatic index of globular proteins. , 1980, Journal of biochemistry.

[26]  Andrej ⩽ali,et al.  Comparative protein modeling by satisfaction of spatial restraints , 1995 .

[27]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[28]  B. Matthews,et al.  Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Lamotte‐Brasseur,et al.  Enzymes from cold-adapted microorganisms. The class C beta-lactamase from the antarctic psychrophile Psychrobacter immobilis A5. , 1997, European journal of biochemistry.

[30]  G. Taylor,et al.  Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. , 1998, Structure.

[31]  T. Yoshimura,et al.  Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization , 1999, Applied and Environmental Microbiology.

[32]  D. Rice,et al.  Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase , 1998, Nature Structural Biology.

[33]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[34]  R. Herbert,et al.  Molecular Biology and Biotechnology of Extremophiles , 1992 .

[35]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[36]  H. Tanaka,et al.  Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)(+)-dependent dehydrogenases. , 1990, Biochemistry.

[37]  C. Gerday,et al.  Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. , 1997, Protein engineering.

[38]  A. Yoshida.  ENZYMIC PROPERTIES OF MALATE DEHYDROGENASE OF BACILLUS SUBTILIS. , 1965, The Journal of biological chemistry.

[39]  J. Janin,et al.  Arginine residues as stabilizing elements in proteins. , 1993, Biochemistry.

[40]  A Sali,et al.  Comparative protein modeling by satisfaction of spatial restraints. , 1996, Molecular medicine today.

[41]  D. Eisenberg,et al.  A method to identify protein sequences that fold into a known three-dimensional structure. , 1991, Science.

[42]  N. Russell,et al.  Physiology and molecular biology of psychrophilic micro-organisms. , 1992 .

[43]  A. Yoshida,et al.  ENZYMATIC PROPERTIES OF ALANINE DEHYDROGENASE OF BACILLUS SUBTILIS. , 1965, Biochimica et biophysica acta.