Identification of critical amino acid residues for human iNOS functional activity.
Nitric oxide (NO) is a short-lived signaling molecule that mediates a variety of biological functions, including vascular homeostasis, neurotransmission, antimicrobial defense and antitumor activities. Three known NOS isoforms (eNOS, nNOS and iNOS) have been cloned and sequenced. Here, we show that upon expression in Escherichia coli using a novel expression vector, an iNOS sequence containing three mutations (A805D, F831S and L832P) within the iNOS reductase domain produced very little functionally active iNOS protein compared to the wild type (wt) iNOS. Each of these point mutations also was individually constructed into the wt iNOS sequence. The activity of the iNOS protein containing the A805D mutation was comparable to wt, while a drastic reduction in iNOS activity was observed for the F831S and L832P mutants. A comparison of the molecular models of the reductase domain of the wt and mutant iNOS revealed a reduced core packing density for the F831S and L832P mutations compared to wt. In addition, the modeling also suggests altered hydrogen bonding, van der Waals and hydrophobic interactions of these mutants.
Naureckiene
Saule
S
Neuroscience Discovery Research, Wyeth Research, CN8000, Princeton, NJ 08543, USA.
Kodangattil
Sreekumar R
SR
Kaftan
Edward J
EJ
Jones
Philip G
PG
Kennedy
Jeffrey D
JD
Rogers
Kathryn E
KE
Chanda
Pranab K
PK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary
D000596:Amino Acids; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II