Pyruvoyl-dependent histidine decarboxylases. Mechanism of cleavage of the proenzyme from Lactobacillus buchneri.

P A Recsei,E E Snell

doi:10.1016/s0021-9258(18)89434-0

Abstract

When Lactobacillus buchneri was grown in the presence of [hydroxyl-18O]serine and pyridoxamine, no 18O was found in its histidine decarboxylase (HisDCase). However, when pyridoxamine was omitted from the growth medium, the labeled serine was incorporated into the HisDCase without dilution. Internal serine residues of the enzyme contained 18O only in their hydroxyl group, while the COOH-terminal serine of the beta chain of HisDCase contained equal amounts of 18O in both its hydroxyl and carboxyl group. This enzyme, like the HisDCase from Lactobacillus 30a (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977), therefore, arises by nonhydrolytic serinolysis of its proenzyme. This result, together with comparative sequence data (Huynh, Q. K., and Snell, E. E. (1985) J. Biol. Chem. 260, 2798-2803), makes it highly probable that all of the pyruvoyl-dependent HisDCases arise by a similar mechanism from inactive proenzymes.

Highlights

When Lactobacillus buchnerwi as grown in the pres-was demonstrated by supplying [hydr~xy-'~O]serineto the ence of[hydr~xyl-'~O]serinaend pyridoxaminen, o "0 growing cultures of Lactobacillus 30aand showing that while was found in its histidine decarboxylase (HisDCase). control serine residues from the a chain of the subsequently
When pyridoxamine was omitted from the isolated HisDCase contained only itnheir hydroxyl growth medium, the labeled serine was incorporated groups, the carboxyl-terminal serine residue from the j3 chain intotheHisDCasewithoutdilution.Internal serine residues of theenzymecontained "0 onlyintheir hydroxyl group, while the COOH-terminal serine of the B chain of HisDCase contained equal amounts of l8O inboth its hydroxylandcarboxylgroup.This enzyme, like theHisDCasefromLactobacillus 30a
Pyruvoyl-dependent HisDCases from three other bacterial species are known (1). proHisDCase has been detected in only one of these, Lactobacillus buchneri (l),the finding (3) that HisDCases from all threeorganisms have the same sequence at thej[3] chain COOH terminus and the same sequence at thea chain NH2 terminus asthat shown for the Lactobacillus 30a enzyme in Equation 1implies that all four enzymes arise from inactive proenzymesthat share a common

Summary

EXPERIMENTAL PROCEDURES

Fractionation and Immobilizationof Rabbit Antibodies to Histidine Decarboxylase-Rabbit antiserum (100ml) to histidine decarboxylase from Lactobacillus 30a wasprepared as described previously (4). Oxygen from H2lS0is not incorporated into the newly gen- fractions obtained by elution of the column with Buffer A were pooled, erated carboxyl terminus of the j3 chain; instead, this oxygen and theprecipitate that formed upon addition of ammonium sulfate is supplied by the hydroxyl group of the serine residue (Ser in Equation1)that becomes the pyruvate residue (Pru)of the active enzyme (2) That this mechanism, initially demonstrated for a mutant proenzyme with a slowed activation rate, pertains for activation of wild-type proHisDCase under growth conditions at 0 'C to 50%of saturation was collectedby centrifugation, dissolved in 15 ml of0.15 M NaCl, and dialyzed against 0.15 M NaCl, 1 mM sodium borate, pH 8.4 (Buffer B) for 24 h at 0 "C. These values were determined from intensities of the molecular ion (M) and thieons at m/z M 15 and M R

RESULTS

DISCUSSION

PUP groupb x