Biochemical Identification And Phylogenetic Analysis Of Haloalkane Dehalogenases In Alcanivorax

Alcanivorax dieselolei B-5T is an important oil degradation bacteria strain isolated and identified by our group and has potential meanings in environmental improvement and biological study. Carbon source experiments showed A. dieselolei B-5can use a variety of halogenated alkanes as sole carbon and energy source for growth.1. Cloning, expression and enzymatic analysis of HLDs in A. dieselolei B-5.We expressed and purified the two putitave HLDs in B-5, DadA and DadB; detected their enzyme activities and substrate specificity, measured the steady-state kinetic constants and the optimum temperature and pH of DadB, checked the stability of DadB in different buffers with different pH by the dynamic light scattering(DLS) methods, discussed the structural basis of DadB’s enzymology characteristics by homology modeling.DadA has the most stringent substrate selectivity in the identified HLDs, its substrate range significantly smaller than other HLDs identified, representing a new subfamily of specificity. Expression and purification of DadB are simple and efficient. DadB has broad substrates range and high activity profile, preferring small substrates (C2and C3), brominated substrates and chloroalkene substrates. As far as known, DadB has the highest systemic activity towards chlorinated, brominated, iodinated C2and C3substrates in all HLDs identified. Steady-state kinetic constants of DadB with6substrates are basiclly in the similar magnitude, while DadB has higher Kcat values with several substrates such as3-chloro-2-methylpropene and4-bromobutanenitrile. Moreover,4-bromobutanenitrile showed distinct substrate self-inhibition phenomenon.DadB has excellent stability of the enzyme acitivity. DadB keeps obvious activity between pH5.0to pH10.0and the peak occurred at pH8.0. DadB has activity from20℃to55℃with the optimum temperature at50℃. DadB aggregates very fast to form large polymers with different size in the buffer has a pH lower than7.5. When the buffer’s pH≥7.5, DadB exists in the form of monomer in solution and keep stable in a long time.Two structural models were obtained and they have typical α/β-hydrolase structural fold, consisting of the core domain and the cap domain. Topological structures of catalytic pentad amino acids in DadA and DadB are very consistent with the HLDs already had the three dimensional structures. But DadB has an open active cavity with greater access tunnel, also takes Ile/Trp couple as the residue barrier to separate the main tunnel and the slot tunnel. This may be an important cause of the preference of DadB for small substrates.2. Phylogenetic analysis of HLDsWe analyzed genomes of37Alcanivorax and found out51HLDs genes in33Alcanivorax. This paper analyzed the phylogeny of putative HLDs from Alcanivorax, proposed that Alcanivorax bacteria have obtained different evolutionary clades of HLDs from the other species in at least4independent evolutionary events occured at different time and subsequently these HLDs spread between different strains of Alcanivorax maybe through horizontal gene transfer.This paper also did the systematic phylogenitic analysis with almost all of HLDs can found now.Through bioinformatics technique,421putative HLDs sequences were found out from non-redundant protein sequences (nr) database of NCBI using17HLDs sequences identified as queries. Phylogenetic analysis with these obtained421HLDs together with53putative HLDs sequenced by our group shows that DadA and other19putative HLDs all from Alcanivorax represents a new evolutionary branch of HLDs. This maybe the reason why DadA has unique enzyme properties of activity. This paper preliminarily analysed the the natural functions and the important role of HLDs in the microorganism and in the environment, proposed that marine environment may be a large genetic resource pool of HLDs sequences and may play an important role in the propagation and evolution process of HLDs.In summary, this study proved for the first time there are HLDs with identified dehalogenation activity existing in Alcanivorax, analyzed the role of these HLDs for Alcanivorax to take halogenated compounds as sole carbon source. This paper obtained and identified two HLDs, DadA with novel substrate selectivity and sequence specificity, DadB with the most wide range of substrates and the highest activity in the knowen HLDs identified untill now to small substrates with serious recalcitrance. This paper expanded the genetic resources of HLDs, offered alternative targets for the practical applications of HLDs, provided research materials for improving catalytic efficiency and substrate selectivity of HLDs by protein engineering technique, and also proved oil degrading bacteria A. dieselolei B-5also has potential application in degradation of halogenated pollutants.Putative HLDs sequences has already been found in almost120genuses. Taking Alcanivorax as a good model, this paper for the first time analyzed the origin, evolution, transferring, spreading in a perticular genus, provided important clues and ideas for discussing the natural functions of HLDs and the possible reason of its spread in so many different genuses and for researching the roles and meanings for metabolism and survival.