Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase.
The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app) for acetyl coenzyme A increased while the Km(app) for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-acetylglutamate at the maximal rate.
Center for Genetic Medicine Research, Children's National Medical Center, Washington D.C., United States of America ; Department of Integrative Systems Biology, The George Washington University, Washington D.C., United States of America.
Ljubica
L
Caldovic
Center for Genetic Medicine Research, Children's National Medical Center, Washington D.C., United States of America ; Molecular and Cellular Biology Program, University of Maryland, College Park, Maryland, United States of America.
Nantaporn
N
Haskins
American Society for Radiation Oncology, Fairfax, Virginia, United States of America.
Amy
A
Mumo
Center for Genetic Medicine Research, Children's National Medical Center, Washington D.C., United States of America.
Himani
H
Majumdar
Department of Biological Sciences, Rowan University, Glassboro, New Jersey, United States of America.
Mary
M
Pinter
Center for Genetic Medicine Research, Children's National Medical Center, Washington D.C., United States of America.
Mendel
M
Tuchman
Department of Biological Sciences, Rowan University, Glassboro, New Jersey, United States of America.
Alison
A
Krufka
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D051046:Amino-Acid N-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000201:enzymology / Q000378:metabolism*; D004795:Enzyme Stability; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization; D058767:Protein Unfolding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D005971:Glutamates; D029961:Zebrafish Proteins; D018698:Glutamic Acid; D000105:Acetyl Coenzyme A; D001120:Arginine; D051046:Amino-Acid N-Acetyltransferase; C016195:N-acetylglutamic acid