TY - JOUR
T1 - The ybcF Gene of Escherichia coli Encodes a Local Orphan Enzyme, Catabolic Carbamate Kinase
AU - Kim, Nam Yeun
AU - Kim, Ok Bin
PY - 2022/12/28
Y1 - 2022/12/28
N2 - Escherichia coli can use allantoin as its sole nitrogen source under anaerobic conditions. The ureidoglycolate produced by double release of ammonia from allantoin can flow into either the glyoxylate shunt or further catabolic transcarbamoylation. Although the former pathway is well studied, the genes of the latter (catabolic) pathway are not known. In the catabolic pathway, ureidoglycolate is finally converted to carbamoyl phosphate (CP) and oxamate, and then CP is dephosphorylated to carbamate by a catabolic carbamate kinase (CK), whereby ATP is formed. We identified the ybcF gene in a gene cluster containing fdrA-ylbE-ylbF-ybcF that is located downstream of the allDCE-operon. Reverse transcription PCR of total mRNA confirmed that the genes fdrA, ylbE, ylbF, and ybcF are co-transcribed. Deletion of ybcF caused only a slight increase in metabolic flow into the glyoxylate pathway, probably because CP was used to de novo synthesize pyrimidine and arginine. The activity of the catabolic CK was analyzed using purified YbcF protein. The Vmax is 1.82 U/mg YbcF for CP and 1.94 U/mg YbcF for ADP, and the KM value is 0.47 mM for CP and 0.43 mM for ADP. With these results, it was experimentally revealed that the ybcF gene of E. coli encodes catabolic CK, which completes anaerobic allantoin degradation through substrate-level phosphorylation. Therefore, we suggest renaming the ybcF gene as allK.
AB - Escherichia coli can use allantoin as its sole nitrogen source under anaerobic conditions. The ureidoglycolate produced by double release of ammonia from allantoin can flow into either the glyoxylate shunt or further catabolic transcarbamoylation. Although the former pathway is well studied, the genes of the latter (catabolic) pathway are not known. In the catabolic pathway, ureidoglycolate is finally converted to carbamoyl phosphate (CP) and oxamate, and then CP is dephosphorylated to carbamate by a catabolic carbamate kinase (CK), whereby ATP is formed. We identified the ybcF gene in a gene cluster containing fdrA-ylbE-ylbF-ybcF that is located downstream of the allDCE-operon. Reverse transcription PCR of total mRNA confirmed that the genes fdrA, ylbE, ylbF, and ybcF are co-transcribed. Deletion of ybcF caused only a slight increase in metabolic flow into the glyoxylate pathway, probably because CP was used to de novo synthesize pyrimidine and arginine. The activity of the catabolic CK was analyzed using purified YbcF protein. The Vmax is 1.82 U/mg YbcF for CP and 1.94 U/mg YbcF for ADP, and the KM value is 0.47 mM for CP and 0.43 mM for ADP. With these results, it was experimentally revealed that the ybcF gene of E. coli encodes catabolic CK, which completes anaerobic allantoin degradation through substrate-level phosphorylation. Therefore, we suggest renaming the ybcF gene as allK.
KW - allantoin
KW - carbamoyl phosphate
KW - catabolic carbamate kinase
KW - ybcF
UR - http://www.scopus.com/inward/record.url?scp=85144638599&partnerID=8YFLogxK
U2 - 10.4014/jmb.2210.10037
DO - 10.4014/jmb.2210.10037
M3 - Article
C2 - 36384810
AN - SCOPUS:85144638599
SN - 1017-7825
VL - 32
SP - 1527
EP - 1536
JO - Journal of Microbiology and Biotechnology
JF - Journal of Microbiology and Biotechnology
IS - 12
ER -