EVIDENCE THAT HETR PROTEIN IS AN UNUSUAL SERINE-TYPE PROTEASE

The hetR gene plays a very important role in cell differentiation of h eterocystous cyanobacteria, To understand the mechanism of the hetR ge ne product in regulation of heterocyst differentiation, the recombinan t HetR protein (rHetR) was overproduced in Escherichia coli, Purified rHetR was unstable and degraded easily in solution, Phenylmethanesulfo nyl fluoride, a serine-type protease inhibitor, prevented the degradat ion and was shown to modify covalently rHetR, Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl fluoride a nd DnsF was at the same site of rHetR and required Ca2+. S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot di fferentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with Dns F, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, an d the labeled peptide was isolated and sequenced, The labeled peptide matches a sequence from HetR, These results show that HetR is a protea se.