No abstract

The D/L ratio of aspartic acid enantiomers in proteins of low turnover is generally accepted as a reliable procedure for age determination. In our study, twelve samples of eyetooth dentin were analyzed for age determination. The pure insoluble collagen isolated from eyetooth dentin was obtained by an EDTA demineralization process. Free amino acids obtained after collagen hydrolysis were converted into o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) derivatives for HPLC analysis under modified conditions and into trifluoroacetic acid isopropyl esters for GC analysis, respectively. The modified HPLC procedure used phosphate buffer and acidified sample matrix prior to injection which resulted in suppression of peak tailing of both diastereomers, thus allowing achievement of both good selectivity and good resolution. To ensure the high accuracy of the developed method the other parameters, i.e. specificity, precision, linearity, LOD, and LOQ, were also determined. Nine collagen samples covering the age range of 18 to 84 years were used for the determination of coefficient of racemization (KR) and calculation of parameters for age estimation. The regression equations for the data set analyzed were as follows: KR= 0.0005 x age + 0.0262 (R2 = 0.9639) for HPLC, and KR= 0.0006 x age + 0.0319 (R2 = 0.9374) for GC, respectively.

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxopropenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the L-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.Keywords: lincomycin; L-DOPA; derivative of muconic acid semialdehyde; 2,3-extradiol dioxygenase; 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid.Lincomycin A and, particularly, its semisynthetic derivative clindamycin belong to clinically important antibiotics, yet, our knowledge of lincomycin biosynthesis is rather fragmentary.Structurally, lincomycin A is composed of two moieties, a sugar moiety 6-amino-6,8-dideoxy-1-thio-D-erythroa-D-galactooctopyranoside (methylthiolincosaminide) and an amino acid derivative 4-n-propyl-L-hygric acid, linked by an amide bond. A pathway leading to 4-n-propyl-L-hygric acid moiety formation was proposed based on determination of the carbon and nitrogen atoms biosynthetic origin using feeding experiments with subsequent NMR analysis [1]. The 4-n-propyl-L-hygric acid moiety is derived from L-tyrosine, which is hydroxylated to yield L-3,4-dihydroxyphenyl alanine (L-DOPA). The subsequent steps include 2,3-extradiol cleavage of the L-DOPA aromatic ring, cyclization to produce the five-membered ring containing nitrogen (Fig. 1), loss of two carbons, C-methylation at the aliphatic side chain and double reduction to yield propylproline. After condensation of the sugar and aglycon moieties into N-demethyllincomycin, the aglycon is N-methylated to yield the 4-n-propyl-L-hygric acid moiety. Although the scheme of the pathway was drawn in 1984, the one and only intermediate of the multistep L-DOPA conversion to propylproline, 1,2,3,6-tetradehydro-propylproline, was identified [2].Sequence analysis of the Streptomyces lincolnensis lincomycin biosynthetic gene cluster revealed 29 ORFs with putative biosynthetic or regulatory (lmb genes) and resistance functions (lmr genes) [3,4]. Assigning functions to the proteins encoded by the lmb genes is considerably complicated by limited information on the intermediates of the lincomycin biosynthetic pathway. The function has been demonstrated experimentally for three of the lmb genes: lmbJ [5], which codes fo...