The StrQ protein encoded in the gene cluster for 5′‐hydroxystreptomycin of Streptomyces glaucescens GLA.0 is a α‐ D‐glucose‐1‐phosphate cytidylyltransferase (CDP‐ D‐glucose synthase)

Abstract

The gene strQ was identified as the last gene of a putative transcription unit, strB1FGHPQ, located in the gene cluster for the production of 5′-hydroxy-streptomycin (OH-Sm) in Streptomyces glaucescens GLA.0. [In contrast, the corresponding operon in the str/sts-gene cluster of the Sm-producer Streptomyces griseus, strB1FGHIK, differs in the two distal genes; Mansouri, K. & Piepersberg, W. (1991) Mol. Gen. Genet. 228, 459Ϫ469.] The deduced StrQ protein exhibited similarities to members of the enzyme family of hexose-1-phosphate nucleotidylyltransferases (NDP-hexose synthases or pyrophosphorylases), with the strongest similarity to the subfamily of A-D-glucose-1-phosphate cytidylyltransferases (CDP-D-glucose synthases). The StrQ protein was heterologously expressed in Escherichia coli. The purified protein revealed an enzyme activity of that of a CDP-D-glucose synthase and a substrate specificity restricted to CTP and A-D-glucose 1-phosphate. The K m and V max values determined for CTP are 44 µM and 920 µM and for A-D-glucose 1-phosphate 195 µM and 1.06 mM, respectively. The CDP-D-glucose synthase activity was also detected in cells of S. glaucescens under the conditions of antibiotic production, but was absent from cells of the streptomycin producer S. griseus N2-3-11. Also, the genomes of several strains of S. griseus did not seem to possess strQ-related genes. In contrast, hybridisation experiments indicated that genes homologous to strQ were probably present in various other actinomycetes producing aminoglycosides. A possible function of the StrQ protein in the OH-Sm biosynthetic pathway of GLA.0 is discussed.

Kinetic and Structural Analysis of α-d-Glucose-1-phosphate Cytidylyltransferase from Salmonella typhi

“…4a. The backbone amide nitrogen of Gly 10 , the carbonyl oxygen of Ser 106 , and the guanidinium group of Arg 111 all lie within hydrogen bonding distance to the carbonyl oxygen, the amino group, and the ring nitrogen, respectively, of the cytosine base. The 2-hydroxyl group of the nucleotide ribose sits within ϳ2.9 Å of the backbone amide nitrogen of Gly 11 and a water molecule, whereas the 3-hydroxyl group lies within 2.9 and 2.7 Å of the backbone nitrogen of Gly 130 and the carbonyl oxygen of Leu 8 , respectively.…”

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…[131,132] Interestingly, a DdhA homologue (StrQ) was found in the streptomycin (124) gene cluster of Streptomyces glaucescens, and its cytidylyltransferase activity was verified in vitro. [133] However, TDP-glucose has been implicated as the precursor of the streptose moiety (26) of streptomycin (see also Scheme 4). [122] Thus, StrQ may participate in the biosynthesis of the Nmethyl-l-glucosamine (125) moiety of streptomycin.…”

“…Deuterium exchange studies have shown that epimerization at C-5 is much more facile than at C-3 and thus likely occurs first. After C-5 epimerization, a ring-flipped intermediate (133) in the 1 C 4 conformation typical of l-sugars is proposed to form in order to avoid steric clashes between the 5-methyl group, the O-1 atom, and His65. Intermediate 133 is likely in equilibrium with a twist-boat conformation (134) in which the C-3 hydrogen atom is orthogonal to the plane of the 4-keto group to facilitate C-3 proton abstraction.…”

Biosynthetic genes for aminoglycoside antibiotics

“…14 However, only a few biosynthetic genes were heterologously expressed and investigated to characterize the function to date. [15][16][17][18] Among those, the characterization of L-glutamine: syllo-inosamine aminotransferase StsC gave important information to elucidate the aminocyclitol-containing aminoglycosides biosynthetic pathway. 15 Using the hybridization method, Piepersberg and co-workers 15 showed that its homologous gene is conserved in the aminocyclitol antibiotic-producing microorganisms.…”