Invertebrate aspartyl/asparaginyl beta-hydroxylase: potential modification of endogenous epidermal growth factor-like modules.
An invertebrate alpha-ketoglutarate-dependent aspartyl/asparaginyl beta-hydroxylase, which posttranslationally hydroxylates specific aspartyl or asparaginyl residues within epidermal growth factor-like modules, was identified, partially purified and characterized. Preparations derived from two insect cell lines catalyzed the hydroxylation of the expected asparaginyl residue within a synthetic epidermal growth factor-like module. This activity was found to be similar to that of the purified mammalian aspartyl/asparaginyl beta-hydroxylase with respect to cofactor requirements, stereochemistry and substrate sequence specificity. Furthermore, recombinant human C1r, expressed in an insect cell-derived baculovirus expression system, was also found to be hydroxylated at the expected asparaginyl residue. Thus, these results establish the potential for invertebrate aspartyl/asparaginyl hydroxylation. Since several invertebrate proteins known to be required for proper embryonic development contain a putative consensus sequence that may be required for hydroxylation, the studies presented here provide the basis for further investigations concerned with identifying hydroxylated invertebrate proteins and determining their physiologic function.
Monkovic
D D
DD
Merck Research Laboratories, West Point, Pennsylvania 19486.
VanDusen
W J
WJ
Petroski
C J
CJ
Garsky
V M
VM
Sardana
M K
MK
Zavodszky
P
P
Stern
A M
AM
Friedman
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015923:Complement C1r / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000201:enzymology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase; D015923:Complement C1r