A series of positively-charged derivatives has been prepared by reaction of histamine with substituted pyrylium salts. These pyridinium histamine derivatives were investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely the human isoforms hCA I, II and VII. Activities from the subnanomolar to the micromolar range were detected for these compounds as activators of the three isoforms, confirming the validity of current and previous designs. The substitution pattern at the pyridinium ring was the main factor influencing activity, the three isoforms showing different structural requirements for good activity, related with the number of pyridinium substituting groups and their nature, among various alkyl, phenyl and para-substituted styryl moieties. We were successful in identifying nanomolar potent and selective activators for each isozyme and also activators with a relatively good activity against all isozymes tested--valuable lead compounds for physiology and pathology studies involving these isozymes.
Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production.
-Salt (I), ob taine d by the Ba yer-Pic ca rd synthe sis, is inve stiga ted as ac tivato r of se vera l cyto so lic a nd tran sme mbra ne c arbo nic a nhyd r a se is of or ms . Na no-to m icrom olar ra nge a ctivities are obs er ve d aga i nst som e ph ysio logical ly re leva nt c a rbonic a nhydra se s. A n X -ra y c rysta l struc tur e of hCA I I in c omple x with (I ) evide nc es a bindin g m ode nev er ob se rved befo r e , with th e trime thylp yridinium r ing pointing tow a rds the m eta l ion dee p within the enz yme ca vity, a s we ll as se ve ra l s t r o ng h y dr op h o bi c i n t e r a c t i ons s tabiliz ing the a dduc t. T his is the firs t exa mple of a carb onic anhy drase a ctiva tor wh ich time-de pen dently bec ome s an in hibitor.
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