Study On The Molecular Mechanisms For The Catabolism Of Red Algal Monosaccharide 3,6-anhydro-L-galactose By Marine Vibrio

Marine algae are major contributors to marine primary productivity.Marine red algae are rich in agarose,carrageenan and other polysaccharides,which have been widely used in agriculture,biomedicine,food,pharmaceutical and energy industries.Marine bacteria play an important role in the degradation and circulation of marine algal polysaccharides.Recently,the bacterial metabolic pathways of 3,6-anhydro-L-galactose(L-AHG),a major component of red algal monosaccharides,have been reported.However,the molecular mechanisms for the utilization of L-AHG by marine bacteria are unclear.In this paper,we have systematically studied the 3,6-anhydro-L-galactose dehydrogenase from marine bacterium Vibrio variabilis JMC19239(VvAHGD for short),which initiates the L-AHG metabolism pathway.The molecular mechanism for the oxidation of L-AHG by VvAHGD was elucidated by biochemical,structural and mutaional analyses.The comparative analyses of sequences,structures and enzyme kinetic parameters showed that VvAHGD and its homologous proteins represent a new family of aldehyde dehydrogenase(ALDH)superfamily,which we named L-AHGDH family.In addition,the crystal structure of the 3,6-anhydro-L-galactonate cycloisomerase from marine bacterium Vibrio sp.EJY3(VejACI)was resolved,which catalyzes the second-step reaction of the L-AHG metabolic pathway.The structure of VejACI was preliminarily analyzed.Our study on the key enzymes involved in the L-AHG metabolic pathways of marine Vibrio is not only helpful for better understanding the role of marine bacteria in the degradation and circulation of the marine algal polysaccharides,but also lays a foundation for better application of algal polysaccharides in the industrial,especially in the development of bio-fuels.1.Molecular mechanism for the oxidation of L-AHG by a 3,6-anhydro-L-galactose dehydrogenase(VvAHGD)The gene encoding a 3,6-anhydro-L-galactose dehydrogenase(VvAHGD)was identified from the genome of marine bacterium Vibrio variabilis JMC19239 by blast analysis.VvAHGD was heterologously expressed in Escherichia coli BL21(DE3)and purified.Biochemical characterization showed that VvAHGD could efficiently oxidize L-AHG with high specificity.The optimum reaction temperature and optimum pH for VvAHGD were 40?and 7.0,respectively.VvAHGD formed stable dimers in solution.Due to the lack of relevant structural information,the molecular mechanism for the catalysis by VvAHGD is not clear.To reveal the catalytic mechanism of VvAHGD,the crystal structures of VvAHGD and the VvAHGD-NADP+complex were resolved at 2.7 A and 2.37 A resolutions,respectively.The structure of VvAHGD-NADP+complex was also modeled with L-AHG.Structurally,monomeric VvAHGD consists of three domains:a catalytic domain,a cofactor binding domain and an oligomeric domain.Based on structural,mutational,and biochemical analyses,the cofactor channel and the substrate channel of VvAHGD were identified and key residues involved in the binding of NADP+and L-AHG and in the catalysis were also revealed.Comparative structural analysis of VvAHGD and VvAHGD-NADP+complex showed that the key catalytic residues Cys282 and Glu248 undergo obvious conformational changes before and after the binding of NADP+molecules.VvAHGD performs catalysis by controlling the consecutive connection and interruption of the cofactor channel and the substrate channel via the conformational changes of its two catalytic residues Cys282 and Glu248.Thus,the possible molecular mechanism of VvAHGD was elucidated.2.The discovery of a new aldehyde dehydrogenase family-the L-AHGDH familyVvAHGD belongs to the aldehyde dehydrogenase(ALDH)superfamily.Although VvAHGD is similar to known ALDHs in sequence and structure,the substrate specificity of VvAHGD is different from those of other ALDHs.The molecular mechanism for the substrate recognition of AHGD is still unclear:Phylogenetic analysis showed that VvAHGD and its homologous sequences form an independent branch in the ALDH superfamily,and different branches in the tree show different substrate specificities.To reveal the mechanism for the substrate recognition of AHGD,a detailed comparative structural analysis of VvAHGD and other ALDHs was carried out.The comparative analysis of the cofactor channels and the substrate channels of ALDHs from different families showed that the cofactor channels are very conservative in the different families of ALDH,and we identified two key amino acid residues that may affect the cofactor specificities of ALDHs.Unlike the cofactor channels,the substrate channels were quite different among ALDHs from different families.The differences in the shape,size,electrostatic surface,and amino acid residue composition of the substrate channels lead to the different substrate preferences of VvAHGD from other ALDHs,which is supported by the different kinetic parameters of VvAHGD against different aldehydes.Based on our results,we proposed that VvAHGD and its homologs represent a new ALDH family,which is named the L-AHGDH family in this study.3.Structural analysis of a 3,6-anhydro-L-galactonate cycloisomerase(VejACI)The 3,6-anhydro-L-galactonate cycloisomerase(VejACI)from marine bacterium Vibrio sp.EJY3 was heterologously expressed in E.coli BL21(DE3)and purified.VejACI catalyzes the second-step reaction in the bacterial degradation pathway of L-AHG.Sequence analysis showed that VejACI is a cyclic isomerase,which belongs to the subfamily of mandelase in the enolase superfamily.VejACI formed stable dimers in solution.We also crystallized the protein of VejACI and solved its crystal structure.Like other enolases,monomeric VejACI consists of a barrel domain and a CAP domain.In VejACI,Lys169 and His300 act as the general acid/base,respectively.The preliminary structural analysis showed that the active site of VejACI contains no metal ions such as Mg2+ and Ca2+,different from the reported enolases which usually depend on metal ions during the catalysis.Whether the activity of VejACI depends on metal ions needs further biochemical verification.Our study on VejACI lays a foundation for further study on the catalytic mechanisms of ACIs in the bacterial degradation pathway of L-AHG.