Kinetic and functional analysis of L-threonine kinase, the PduX enzyme of Salmonella enterica.
The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His(8)-PduX) was produced in Escherichiacoli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.
Fan
Chenguang
C
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Fromm
Herbert J
HJ
Bobik
Thomas A
TA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D020816:Amino Acid Motifs; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D019779:Salmonella enterica / Q000201:enzymology*; D016415:Sequence Alignment; D013912:Threonine / Q000031:analogs & derivatives / Q000378:metabolism
D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D013912:Threonine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases