Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation.

The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.

Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

Functional analysis of norcoclaurine synthase in Coptis japonica.

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.

Purification, crystallization and preliminary X-ray diffraction data from selenomethionine glycinamide ribonucleotide synthetase.

In this study, the overexpression, purification and crystallization of selenomethionine (SeMet) incorporated glycinamide ribonucleotide synthetase (GAR-syn) from Escherichia coli are reported. The overexpression of SeMet GAR-syn was placed under the control of the isopropylthio-beta-galactoside (IPTG) inducible T7 RNA-polymerase system. The newly developed construct contained a removable histidine tag on the amino terminus of GAR-syn, which allowed rapid purification using metal-chelate chromatography techniques. The SeMet GAR-syn crystals were grown by hanging-drop vapor diffusion and belong to the space group P212121 with unit-cell parameters a = 56.2, b = 62.4 and c = 129.8 A and a single monomer in the asymmetric unit. The crystals diffract to 1.6 A resolution and have led to the determination of multiple-wavelength anomalous diffraction phases to 2.2 A resolution.