Study On The Biosynthesis Mechanism Of Trans 4-n-propylproline Units In Lincomycin A Molecule

Many important clinical drugs and active molecules are natural products or their derivatives.Lincomycin?often referring to the A component?,a widely used antibiotic,is mainly used for the treatment of the infection caused by gram-positive bacteria.Structurally,lincomycin consists of two units,an eight-carbon unit?methylthiolincosamide,MTL?and a unique amino acid residue?trans-4-propyl-L-proline,PPL?,which are connected via an amide bond.In recent years,great progress has been made in the biosynthesis of lincomycin.For example,low-molecular-weight thiols were found to be involved in the construction of the lincomycin backbone through two unusual S-glycosylations.Notably,this is the first paradigm for EGT-associated biochemical process and for the poorly understood MSH-dependent biotransformations,a newly described model that is potentially common in the incorporation of sulfur.During the biosynthesis of the PPL unit,the fragmentation of the terminal two-carbon unit that involves the activity of the Ntn hydrolase superfamily protein,LmbA,is extremely rare.Although much progress has been made,there are still ambiguities in the biosynthetic passway.In this study,we focus on the genes related to the formation of PPL unit in the biosynthesis of lincomycin by combining both in vivo and vitro approaches.An analysis of the biosynthesis gene cluster of lincomycin revealed that the genes lmbA,lmbB1,lmbB2,lmb W,lmbX and lmbY might participate in PPL formation.Among them,the first four proteins have been characterized previously,whereas the functions of lmbX and lmbY remain to be determined.By constructing related plasmids,lmbX and lmbY were deleted individually in S.lincolnensis,resulting in the corresponding mutant strains that were able to produce lincomycin analogs.The isolation and characterization of these lincomycin analogues from corresponding mutants led to the hypothesis that LmbY is related to the reduction of the unsaturated bond in PPL formation,and that LmbX is involved in the chirality at the 4-position of the PPL unit.Feeding intermediates that were chemically synthesized into the cultures of?lmbA&lmbA2991 mutant strain can restore the production of lincomycin A.bioinformatics analysis revealed that LmbY belongs to the Bacteria luciferase superfamily and is a F₄₂₀-dependent reductase.This enzyme catalyzes the reduction of double bonds in the pyrrole ring of the PPL unit based on in vitro enzymatic assays.The substrate used in LmbY activity assays can be a PPL biosynthetic intermediate in an enamine form or a chemically synthesized compound in an imine form.In both cases,the double bond in the pyrrole ringcan be catalytically reduced,with a difference in the position of the remaining double bond that resides within the n-propyl side chain of the resulting product.LmbX belongs to the diaminopimelic acid epimerase superfamily.Based on both in vivo knockout experiments and in vitro enzymatic assays,LmbX was characterized to catalyze the change in the positions of the two double bonds of the PPL unit.