Covalent structure of subunits of bacterial luciferase: NH2-terminal sequence demonstrates subunit homology.

Abstract

The heterodimeric subunit structure of bacterial luciferase was'demonstrated more than 10 years ago. The enzymes from both Beneckea harveyi and Photobacterium fischeri have since been studied in detail; they each consist of two nonidentical subunits, designated a and f. Both are required for bioluminescence activity, with the active center apparently confined to the a subunit. Amino acid sequence analysis-of the NH2 termini of the a and P subunits of the B. harveyi and P. fischeri luciferases not only confirms the earlier observation that the a subunits are homologous but also demonstrates that the NHrterminal sequences of the P subunits-of the luciferases from the-two genera are homologous. Furthermore, within each luciferase, the NHrterminal sequences of the a and 13 subunits are similar, suggesting the possibility that the genes coding for a and ft may have arisen by gene duplication, presumably prior to divergence of the lines leading to present-day luminous bacteria.Bacterial luciferase catalyzes the mixed function oxidation of reduced flavin mononucleotide (FMNHI2) and a long-chain saturated aldehyde to yield the corresponding carboxylic acid, oxidized flavin, and blue-green light (Xmaha t490 nm) (2). The enzyme is a heterodimer (a/3) with subunit molecular weights obtained from electrophoresis in sodium dodecyl sulfate of 42,000 and 37,000 for Beneckea harveys a and 3, respectively, and 41,000 and 38,000 for the corresponding Photobacteorum fischeri subunits (3) ¶1-The structural nonidentity of the luciferase a'and 3 subunits was first demonstrated clearly by separation of the subunits on columns of DEAE-cellulose in urea-containing buffers (5) and by electrophoretic resolution in polyacrylamide gels containing urea or sodium dodecyl sulfate (3). The a''and /3 subunits have been shown to have different amino acid compositions (3) and tryptic peptide maps (4). However, the discovery of three identical tryptic peptides between the higher molecular weight subunits, from the P. fischeri and B. harveyi luciferases suggested that the a subunits of the luciferases from these two genera are homologous (4).The functional nonidentity of the luciferase a and /3 subunits was first demonstrated by chemical modification of the enzyme (6, 7) and has been confirmed by mutant enzyme analysis (8), additional chemical modification studies (9, 10), differences in susceptibility to proteases (11), and numerous ligand binding studies (12)(13)(14)(15)(16)(17). The enzyme has a single flavin-binding site. The binding of reduced flavin has been demonstrated by using a kinetic technique (12), fluorescence quenching (13), and circular dichroism (14). The binding of oxidized flavin has been demonstrated by equilibrium dialysis (15) (14).The rather compelling conclusion drawn from all of these observations is that the luciferase a and /3 subunits are quite distinct, both structurally and functionally. We

The 1.5-Å Resolution Crystal Structure of Bacterial Luciferase in Low Salt Conditions

“…Structural Similarities-There is extensive structural conservation between the ␣ and ␤ subunits confirming their common origin (4). The topology of the ␣ and ␤ subunits is identical, and the secondary structural elements align exactly with the sequence (Fig.…”

“…All bacterial luciferases studied so far appear to be homologous, and all catalyze the same reaction: The reaction proceeds through a series of intermediates leading to the formation of a C4a hydroxyflavin (for review see Ref. 4). Light emission apparently occurs from this hydroxyflavin, which dehydrates to yield FMN.…”

“…Bacterial luciferase is a heterodimeric enzyme of 77 kDa, composed of ␣ and ␤ subunits with molecular masses of 40 and 37 kDa, and in the case of Vibrio harveyi, 355 and 324 residues, respectively. The two polypeptides, encoded on adjacent genes, luxA and luxB in the lux operon, display sequence homology and appear to have arisen by gene duplication (4). There is a single active center in the luciferase heterodimer that resides on the ␣ subunit (5) and binds one reduced flavin molecule (6,7).…”

ORGANIZATION and REGULATION OF BACTERIAL LUMINESCENCE GENES

“…harveyi than to the p subunit of P. phosphoreum (Cohn et al, 1985;Johnston et ul., 1986;Soly et al, 1988). These similarities suggest that IuxF arose by gene duplication of luxB , as has been proposed for luxA and luxB (Baldwin et al, 1979;Foran and Brown, 1988). With regard to the function of LuxF, the N-terminal sequences of recently described non-fluorescent flavoproteins from P. phosphoreum and P. leiognathi (O'Kane ef al., 1987;Kasai et a/., 1987) are strikingly similar to the first 35 amino acid residues of these polypeptides, so luxF quite probably encodes a non-fluorescent flavoprotein Mancini et a/., 1989;Baldwin et al, 1989).…”

Mathematical model of the Lux luminescence system in the terrestrial bacterium Photorhabdus luminescens

“…It has been suggested that the duplication may have arisen prior to the divergence of the lines leading to present-day luminescent bacteria. 10 The active site is thought to be on the a subunit. The LuxAB enzyme catalyses the oxidation of FMNH 2 (reduced flavin) and a long chain fatty aldehyde to oxidised flavin (FMN) and a long chain fatty acid respectively.…”