Octavian Barzu

Conversion of Methionine to Cysteine in Bacillus subtilis and Its Regulation

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine beta-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine gamma-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an DeltayrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a DeltayrhA DeltacysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.

CMP Kinase from Escherichia coli Is Structurally Related to Other Nucleoside Monophosphate Kinases

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6, have unit cell parameters a = b = 82.3 Å and c = 60.7 Å, and diffract x-rays to a 1.9 Å resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

Regulation of Expression of the 2-Deoxy-d-Ribose Utilization Regulon, deoQKPX, from Salmonella enterica Serovar Typhimurium

Salmonella enterica, in contrast to Escherichia coli K12, can use 2-deoxy-D-ribose as the sole carbon source. The genetic determinants for this capacity in S. enterica serovar Typhimurium include four genes, of which three, deoK, deoP, and deoX, constitute an operon. The fourth, deoQ, is transcribed in the opposite direction. The deoK gene encodes deoxyribokinase. In silico analyses indicated that deoP encodes a permease and deoQ encodes a regulatory protein of the deoR family. The deoX gene product showed no match to known proteins in the databases. Deletion analyses showed that both a functional deoP gene and a functional deoX gene were required for optimal utilization of deoxyribose. Using gene fusion technology, we observed that deoQ and the deoKPX operon were transcribed from divergent promoters located in the 324-bp intercistronic region between deoQ and deoK. The deoKPX promoter was 10-fold stronger than the deoQ promoter, and expression was negatively regulated by DeoQ as well as by DeoR, the repressor of the deoxynucleoside catabolism operon. Transcription of deoKPX but not of deoQ was regulated by catabolite repression. Primer extension analysis identified the transcriptional start points of both promoters and showed that induction by deoxyribose occurred at the level of transcription initiation. Gel retardation experiments with purified DeoQ illustrated that it binds independently to tandem operator sites within the deoQ and deoK promoter regions with K(d) values of 54 and 2.4 nM, respectively.

Fine differences in structure and stability of bacterial adenylate kinases as revealed by IR difference spectroscopy

Changes in the protein backbone conformation and the microenvironment of some amino acids -- induced by point mutations in bacterial adenylate kinases -- were investigated by IR difference spectroscopy in conjunction with temperature and hydrogen-deuterium exchange measurements also monitored by IR.