Sl4 crystallography of enzymes

J. Dohnálek, T. Skálová, T. Kova3⁄4, J. Dušková, P. Kolenko, K. Fejfarová, J. Stránský, L. Švecová, M. Trundová, J. Hašek In sti tute of Bio tech nol ogy, v. v. i. AS CR, Vídeòská 1083, 14220 Praha 4 In sti tute of Macromolecular Chem is try, v. v. i., AS CR, Heyrovského nám. 2, 16206 Praha 6 dohnalek@ibt.cas.cz Knowl edge of three-di men sional struc ture of en zymes plays an im por tant role in our un der stand ing their func tion and is re quired for de sign and mod i fi ca tion of en zy matic prop er ties and sta bil ity. In re cent years we have worked with sev eral en zy matic sys tems where struc tural stud ies pro vided very im por tant in sights into the en zyme be hav iour, its clas si fi ca tion, and helped dis cov ery of pre vi ously not known ac tiv ity or un cov ered struc tural changes upon ligand bind ing. In struc tural stud ies of b-galactosidase from Arthrobacter sp. C2-2 a unique way of ar range ment of the mol e cules in func tional hexamers was dis cov ered. The func tional form of the en zyme re vealed by its crys tal struc ture has con se quences for the ac tual sub strate and prod uct lo gis tics. The sphere-like hexamers form a large cav ity in side the clus ter with three types of chan nels con nect ing it with ex te rior. The six ac tive sites of the hexamer are open into the in ter nal cav ity and are not ac ces si ble from the out side. Thus any sub strate or ligand must pass through the chan nels of the hexamer to reach the cat a lytic site. In fur ther stud ies it was con firmed that lig ands can in deed ac cess the ac tive sites in side the cav ity via the ex ist ing chan nels. Also pres ence of the so called shal low bind ing mode of this glycosyl hydrolase was con firmed by ob ser va tion of in hib i tor bind ing in this 660 kDa struc ture [1]. Struc tural ar range ment and sta bi li za tion fea tures un cov ered by crys tal struc ture of the small laccase from Streptomyces coelicolor for the first time proved ex is tence of the trimerization-de pend ent laccase, in which qua ter nary or ga ni za tion in an in ter est ing way makes the sec ond do main of laccase re dun dant and en sures ex treme sta bil ity [2]. Here, an ex cep tion ally high sol vent con tent value of 83% was ob served. The high sol vent con tent was in ter est ing from the point of view of meth od ol ogy of struc ture so lu tion as it en abled very ef fec tive ap pli ca tion of sol vent flat ten ing to the ini tial phases ac quired from a MAD ex per i ment on cop per ions. Fur ther stud ies of ligand bind ing led to vari a tion of en zyme ar range ment in the crys tal and im proved qual ity of data con nected with ferro cyanide bind ing (act ing as an elec tron do nor). The struc ture so lu tion re vealed also pres ence of a cen tral chan nel which can serve as a route for ac cess to the trinuclear cop per clus ter. Its role still re mains un ex plained. Struc ture of the plant nuclease TBN1 with con firmed anticancerogenic prop er ties opened up many top ics re gard ing non-spec i fic ity of this en zyme ca pa ble of deg ra da tion of ss and ds DNA and RNA. Struc tural sim i lar ity to an other en zyme led to con fir ma tion of phospholipase C-like ac tiv ity and ini ti ated other in ves ti ga tions [3]. Es pe cially map ping of the mo lec u lar sur face electrostatics and dif fer ences be tween sin gle strand and dou ble strand pro cess ing nu cleases of this type sug gest some amino acid res i dues be ing re spon si ble for such spec i fic ity. Struc tural stud ies also brought in sights into ag gre ga tion/dimer for ma tion of this en zyme, which for the first time raises ques tions about spe cific pep tide bind ing in a nuclease ac tive site. Struc tural study of a bac te rial organophosphorous acid anhydrolase pro vided for the first time a com plete view of the en zyme and proved that the en zyme classes of prolidase and OPAA are ba si cally iden ti cal [4]. Ac cess to the ac tive site dif fers in the hu man and bac te rial en zymes al though de pend ence on man ga nese ions re mains con served. The solved struc tures showed de tails im por tant for en zyme dimerization, which is re quired for func tion. Co va lent mod i fi ca tion of the pep tide chain of the bac te rial OPAA was ob served, which was iden ti fied as nickel form ing co va lent bonds to the main chain ni tro gen at oms. X-dif frac tion anal y sis of en zymes brings es sen tial in for ma tion di rectly re lated to func tion of the stud ied sys tems. The most out stand ing are in for ma tion about oligomerization and its ex act mech a nism, po si tion ing and ex act func tion of the cat a lytic amino ac ids, ligand and metal bind ing, and mech a nisms of struc tural sta bi li za tion. Solid re sults of struc tural anal y sis pave route to fur ther mod i fi ca tions of the en zy matic sys tems and uti li za tion in bio tech no log i cal and med i cal ap pli ca tions.