In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N
The biosynthesis of methylamine dehydrogenase (MADH) fromParacoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauBand mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans,mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroidesand introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the nativemau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate thatmauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of themauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed inE. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility ofR. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.
The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCRbased methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1 -K6)-SHMT and des-(A1 -W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1 -V30)-SHMT and des-(A1 -L49)-SHMT were expressed at very low levels, whereas des-(A1 -R58)-SHMT, des-(A1 -G75)-SHMT, des-(Q435 -F483)-SHMT and des-(L299 -F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1 -K6)-SHMT and des-(A1 -W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolateindependent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55"C, the des-(Al-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1 -K6)-SHMT retained 85 % and 70% activity, respectively. Thermal denaturation studies showed that des-(A1 -W14)-SHMT had a lower apparent melting temperature (52 "C) compared to rSHMT (56°C) and des-(A1 -K6)-SHMT (55 "C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2-CO.l M) in the case of des-(Al-W14)-SHMT compared to rSHMT (1.8?0.1 M) and des-(Al-K6)-SHMT (1.7-C0.1 M). The apoenzyme of des-(Al-W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(Al-K6)-SHMT were a mixture of tetramers (~7 5 % and =65%, respectively) and dimers. While, rSHMT and des-(A1 -K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96 % and 94 % enzyme activity, respectively, des-(A1 -W14)-SHMT apoenzyme could be reconstituted only upto 22 %. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.
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