Hua-Shan Huang

Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus.

The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.

Purification and characterization of thermostable glycerol kinase from Thermus flavus

Abstract Glycerol kinase (EC 2.7.1.30; ATP: glycerol 3-phosphotransferase) was purified from Thermus flavus, by ammonium sulfate fractionation and sequential chromatographies on Toyopearl HW65C and DEAE-Toyopearl columns, with an activity recovery of 22.7%. The enzyme is most active at pHs of 9.0 to 9.5. The optimum temperature for the enzyme is 50–70°C. About 50% of the initial activity remains after incubation at 68°C and pH 7.5 for 30 min. The isoelectric point of the enzyme is 4.3. Its molecular weight is estimated to be 220,000 Da by gel filtration on FPLC-Hiload Superdex 200 pg and 58,000 Da by SDS-PAGE, suggesting that it is a tetramer. The activity of the enzyme is completely inhibited by PCMB, HgCl2 and Mn2+. The Km values of the enzyme for glycerol and ATP are 3.8 × 10−5 M and 1.62 × 10−4 M, respectively. The N-terminal amino acid sequence of the enzyme is MNQYMLAIDQGTTSSR.

THERMOSTABLE GLYCEROL KINASE FROM THERMUS FLAVUS : CLONING, SEQUENCING, AND EXPRESSION OF THE ENZYME GENE

The thermostable glycerol kinase (EC 2.7.1.30) gene from Thermus flavus was cloned and expressed in Escherichia coli DH5 alpha. An open reading frame of 1488 bp for the glycerol kinase gene (glpK) starting with an ATG methionine codon was found, which encodes a protein of 496 amino acid residues whose calculated molecular weight is 54,835. The amino acid sequence of T. flavus glycerol kinase is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli. Transformants of E. coli DH5 alpha harboring plasmid pGYK12 with a 1505 bp chromosomal DNA fragment containing the T. flavus glycerol kinase gene showed about 23.8-fold higher glycerol kinase activity than T. flavus.

The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O···H-N:Inh and Gln71:N-H···OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. Thek cat value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k cat/K m ) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an “induced fit” mechanism.

Novel Aminopeptidase Specific for Glycine from Actinomucor elegans

Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly>>Ala>>Met>Arg>Ser>Leu. The K m value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40°C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.

Preliminary crystallographic study of Thermus aquaticus glycerol kinase

Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kalpha rotating-anode X-ray source.

Cloning and Sequencing of the Thermus aquaticus Glycerol Facilitator Gene

The gene glpK, encoding glycerol kinase of Thermus aquaticus has been identified [ Biosci. Biotechnol. Biochem., 62 (1998) 2375-2381]. In the present work, the nucleotide sequence of glpFK operon and the gene glpF encoding glycerol facilitator were determined. T. aquaticus GlpF was predicted to contain 272 amino acids with six putative transmembrane segments and two half-membrane-spanning segments that contained the motif Asn-Pro-Ala, respectively. The amino acid residues involved in the discrimination of glycerol were deduced to be Trp44, Tyr182, and Arg188.