Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4348-4352, July 1984 Biochemistry An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme (lipid activation/lipid binding/hydrophobic site/conformational change/proteolytic activation) YING-YING CHANG AND JOHN E. CRONAN, JR. Department of Microbiology, University of Illinois, 131 Burrill Hall, 407 South Goodwin, Urbana, IL 61801 Communicated by Ralph S. Wolfe, April 4, 1984 ABSTRACT The pyruvate oxidase (pyruvate:ferricyto- lipid activation of an enzyme occurs in vivo and is physiolog- chrome b, oxidoreductase, EC 1.2.2.2) of Escherichia coli is ically significant. markedly activated by phospholipids in vitro. To test the phys- iological relevance of this activation, we isolated an E. coil mu- MATERIALS AND METHODS tant producing an oxidase that is deficient in activation by (and binding to) phospholipids. The mutant oxidase could be Bacterial Strains, Media, and Extract Preparation. Strains fully activated by a specific proteolytic cleavage, indicating CY265 (pox') and YYC124 (poxB3) are isogenic derivatives that the catalytic site is normal. The mutant enzyme functions (4) of E. coli K-12 that carry a deletion of the aceEF (pyru- poorly in vivo, indicating that activation of the oxidase by vate dehydrogenase) genes. Strain YYC124 is a pyruvate ox- phospholipids plays an important physiological role. idase mutant derived from strain CY265 (4). The strains were grown in broth containing 10 mM sodium acetate at 330C, Enzyme activation by membrane phospholipids in vitro has and cell extracts were prepared as described (3, 4). In some often been observed (1, 2) and is generally ascribed a physio- cases, the cell-free extract was heated to 600C for 5 min and logical role. However, it has not been directly demonstrated then centrifuged for 2 min in an Eppendorf centrifuge (this that the lipid activation of any enzyme observed in vitro oc- treatment removed =75% of the endogenous phospholipids curs in vivo and plays a physiological role (1, 2). We have and about one-half of the protein). The oxidase is stable to begun a genetic study (3, 4) of a lipid-activated enzyme of this heat treatment (10, 11). Membrane vesicles were pre- Escherichia coli, pyruvate oxidase (pyruvate:ferricyto- pared as described by Koland et al. (12). chrome b, oxidoreductase, EC 1.2.2.2), to ascertain the rele- Purification of Pyruvate Oxidase. The strains were grown vance of the striking lipid activation of the enzyme observed in a heavily buffered rich broth medium supplemented with in vitro (5, 6) to the' physiological function of the enzyme. glycerol and 10 mM sodium acetate. Two preparations of the E. coli pyruvate oxidase is one of the most thoroughly mutant oxidase were purified from strain YYC124. The first characterized lipid-activated enzymes (1, 5, 6). The enzyme, was done with P. Porter by the method of O'Brien et al. (10) a homotetramer of a Mr 60,000 subunit, catalyzes the con- with omission ofthe affinity column and addition of a second version of pyruvate to acetate and CO2 (5, 6). Each subunit DEAE-Sepharose column step followed by velocity centrifu- carries a tightly bound molecule of flavin and a loosely gation (48 hr at 28,000 x g in a Beckman SW28 rotor) on a bound molecule of thiamine pyrophosphate (TPP), the bind- 10%-40% (wt/vol) glycerol gradient prepared in 0.1 M sodi- ing of which requires Mg2 . In the presence of pyruvate, um phosphate (pH 5.8). The second purification was done TPP, and Mg2+, the addition of any of a large variety of with M. Recny by the procedure of Recny and Hager (11). phospholipids, neutral lipids, or synthetic detergents results The wild-type enzyme was purified (10) by P. Porter from in a dramatic stimulation of activity; the maximum velocity strain CG6, an oxidase-overproducing strain constructed by increases about 20-fold, and the concentration of pyruvate C. Grabau of this laboratory. All three enzyme preparations needed to saturate the enzyme is decreased by a factor of 10 were >90% homogeneous as assayed on NaDodSO4/poly- (5, 6). Activation is accompanied by a tight association of the acrylamide gels or by absorption spectra, and they had spe- lipid activator with the protein. The binding of lipid to the cific activities in the presence of 20 ,M NaDodSO4 of 60-86 enzyme is accompanied by alteration of various properties of x 103 units per mg of protein. It should be noted that we the protein, which suggests conformational changes (5, 6). have studied the oxidase of E. coli K-12, whereas the previ- The lipid activation of pyruvate oxidase can be mimicked ous workers studied the enzyme from E. coli W. However, by a specific limited proteolysis with any of a variety of pro- our work and recent work from the laboratories ofR. Gennis teases in the presence of substrate and TPP (7-9). Under and L. Hager (personal communications) indicate that the K- these conditions, the Mr 60,000 subunit of the tetramer is 12 and the W enzymes are indistinguishable both physically converted to a Mr 58,000 tetrameric species that cannot be and chemically. further activated by (or bind) lipid (7-9). Proteolytic treat- The oxidase was assayed spectrophotometrically using ment in the absence of TPP and pyruvate results in enzyme Na3Fe(CN)6 as the electron acceptor (4, 7-9). Activation inactivation and cleavage to a Mr 51,000 subunit (7-9). was assayed after incubating the enzyme with pyruvate, We have recently demonstrated that the poxB gene of E. TPP, and Mg2+ and the activator for 30 min at room tem- coli encodes pyruvate oxidase (4). Crude extracts of one of perature. A unit of enzyme activity is 1 nmol of pyruvate the poxB mutants studied, poxB3, contained a normal level decarboxylated per min. The in vivo oxidation pf pyruvate of pyruvate oxidase antigen but only 15% of the normal level was assayed using early stationary-phase cells of strains of enzyme activity (4). In this paper, we show that this mu- YYC63 and YYC164, which are pfl pps derivatives 'of tant protein has a fully functional catalytic site but is inactive strains CY265 and YYC124, respectively (4). These addi- because of an alteration in lipid binding. This is evidence that tional mutations were needed to block other pyruvate-utiliz- ing reactions (3, 4). Washed cells previously grown in succi- nate/10 mM acetate/10 mM pyruvate minimal medium (4) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: TPP, thiamine pyrophosphate. 4348 Downloaded by guest on October 2, 2021 Biochemistry: Chang and Cronan Proc. NatL Acad. Sci. USA 81 (1984) 4349 Table 1. Activity of wild-type and mutant oxidase under various conditions Lipid-depleted Crude extract, units extract,* units per mg per mg of protein of protein Additions Wild type Mutant Wild type Mutant None 108 21 16(183)t 4(27)t Chymotrypsin (5 ug/ml) 108 46 33 21 Chymotrypsin (10 tg/ml) 46 196 167 Chymotrypsin (20 ug/ml) 89 1% 167 Triton X-100 (1%) 256 16 Triton X-100 (1%)/chymotrypsin (10 ,g/ml) 167 *These extracts were depleted of endogenous lipid by heating followed by centrifugation. tValues in parentheses are the oxidase activities present before the heat treatment. were assayed for release of 14CO2 from [1-14C]pyruvate, es- Crude extracts of poxB3 strains contained only 15%-20% sentially as described for cell extracts (3). of the normal level of pyruvate oxidase activity, although a Electrophoresis. NaDodSO4/polyacrylamide gel electro- normal amount of oxidase protein having a normal molecular phoresis on 10% acrylamide gels was carried out as de- weight was present (4). We found that addition of chymo- scribed (4, 7, 8). Electrophoretic transfer to nitrocellulose trypsin to these crude extracts (supplemented with pyruvate, for immunoblotting was done at 0.2 A, as described by Bur- TPP, and Mg2+) resulted in a 2- to 4-fold increase in activity, nette (13) with the inclusion of 0.1% NaDodSO4 in the trans- whereas chymotrypsin treatment of extracts of wild-type fer buffer. In addition, Triton X-100 and 10% fetal bovine cells gave no increase in activity (Table 1). Moreover, im- serum were used in place of the Nonidet P-40 and bovine munoblotting these crude extracts with antibody raised serum albumin used by Burnette (13). The anti-pyruvate oxi- against purified pyruvate oxidase showed proteolytic clip- dase concentration was 0.33 mg/ml and 125I-labeled protein ping of the mutant enzyme to the Mr 58,000 subunit charac- A (the gift of R. Kranz) was added to 5 x 104 cpm/ml. teristic of proteolytic activation, whereas the wild-type en- zyme remained largely unclipped (Fig. 1). The result ob- RESULTS tained with the wild-type enzyme was expected, because Intact cells of strain YYC164 (poxB3) were previously endogenous phospholipid was present in the crude extracts, shown to be deficient in pyruvate oxidase activity by the which would activate the oxidase and also partially protect inability of growing colonies to reduce a tetrazolium indica- the enzyme from proteolysis. The contrasting behavior of tor (4). We have now quantitated this in vivo defect by mea- the mutant enzyme strongly suggested that the poxB3 oxi- suring the production of 14Co2 from [1-14C]pyruvate by in- dase was deficient in lipid binding, because the endogenous tact cells of pox' and poxB3 strains blocked in other pyru- lipid neither fully activated the enzyme nor protected the vate-utilizing pathways.