Cloning, expression, and sequence analysis of diaminopropionate ammonia lyase gene from a nonvirulent Salmonella typhimurium PU011.
BACKGROUND
Seeds ofLathyrus sativus, a legume plant, contain 3-oxalyl and 2,3-dioxalyl DAP (O-DAP), neurotoxins which when consumed causes Neurolathyrism or Osteolathyrism, in humans, affecting nervous system and bone formation respectively. Some microorganisms viz virulent and non-virulentSalmonella typhimurium, Salmonella typhi and Pseudomonad have been shown to detoxifyL-alpha,beta-diaminopropionate (DAP), the immediate precursor of O-DAP.


RESULTS
The gene coding for diaminopropionate ammonia lyase (DAPAL) which detoxifies DAP was cloned from nonvirulentS. typhimurium PU011 into Escherichia coli DH5alpha and the nucleotides sequenced (1212 bp). Whereas the specific enzyme activity of DAPAL obtained from recombinantE. coli PU018 was 0.346 U/mg, the specific activity of the enzyme from nonvirulentS. typhimurium PU011 was 0.351 U/mg. The DAPAL corresponding to 43 kDa protein was found both in nonvirulentS. typhimurium PU011 andE. coli PU018. The Km value was found to be 0.740 mM and 0.680 mM forS. typhimurium PU011 and 0.741 mM and 0.683 mM forE. coli PU018 when grown in minimal medium (MM+DAP) andL. sativus seed extracts respectively, indicating that both of them were capable of utilizing the neurotoxins present inL. sativus seeds. The biomass, enzyme production and the effect of pH and temperature on DAPAL enzyme activity from both non-virulentS. typhimurium PU011 andE. coli PU018 were found to be similar.


CONCLUSIONS
The recombinantE. coli PU018 as well as non-virulentS. typhimurium PU011 are as good as pathogenicS. typhimurium in detoxifying DAP, the immediate precursor of O-DAP present inL. sativus seeds.
Rupesh
K R
KR
Department of Biotechnology, Pondicherry University, Kalapet, Pondicherry, 605014, India.
Padma
K R
KR
Saravanan
P
P
Jayachandran
S
S
D000595:Amino Acid Sequence; D000642:Ammonia-Lyases / Q000235:genetics*; D001483:Base Sequence; D018533:Biomass; D003001:Cloning, Molecular; D016000:Cluster Analysis; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009498:Neurotoxins; D011994:Recombinant Proteins / Q000737:chemistry; D012475:Salmonella / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D009498:Neurotoxins; D011994:Recombinant Proteins; D000642:Ammonia-Lyases; C023147:diaminopropionate ammonia-lyase