The structure of the Escherichia coli hemB gene

Li, Jian Ming; Russell, C.S.; Cosloy, Sharon D.

doi:10.1016/0378-1119(89)90394-6

Cited by 51 publications

(42 citation statements)

References 20 publications

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“…The deduced amino acid sequence of B. subtilis hemB is very similar to the sequence of PBG synthase from human (57), rat (11), yeast (40) and E. coli (15,31) (Fig. 6).…”

Section: 1mentioning

confidence: 65%

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III

et al. 1991

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We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

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“…The deduced amino acid sequence of B. subtilis hemB is very similar to the sequence of PBG synthase from human (57), rat (11), yeast (40) and E. coli (15,31) (Fig. 6).…”

Section: 1mentioning

confidence: 65%

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III

et al. 1991

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“…The sequence agreed with the hemB gene sequence derived from the 6Ј-8Ј region of the E. coli genome (GenBank TM accession number U73857). There are significant differences between these and earlier published hemB gene sequences (22,23). Mutations to human PBGS were obtained using the QwikChange technology of Stratagene on the plasmid pMVhum as described previously (4).…”

Section: Methodsmentioning

confidence: 99%

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

Mitchell¹,

Volin

Martins

et al. 2001

Journal of Biological Chemistry

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Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. 13C NMR studies of [3,5-13 C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.Porphobilinogen synthase (PBGS, 1 also known as 5-aminolevulinate dehydratase) is a metalloenzyme that catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen, the monopyrrole precursor of tetrapyrroles (see Fig. 1). Although PBGS proteins from different organisms differ in their use of metal ions, there is remarkable sequence conservation between the PBGS from eubacteria, archaea, and eucaryotes implying a commonality in the overall protein architecture and reaction mechanism (1). The two PBGS studied herein, those of Escherichia coli and human, both require a catalytic Zn(II), neither require monovalent cations, and the E. coli PBGS activity is stimulated by an allosteric Mg(II) (2-5). The overall protein sequence identity between E. coli and human PBGS is 42%, and the active site residues are significantly more conserved.Considerable effort has been applied to the characterization of PBGS proteins from various organisms, and several crystal structures are now available (6 -10). Yet much of the chemical reaction mechanism remains speculative (7). Only one intermediate, an enzyme-substrate Schiff base, has been identified (11). Several x-ray crystal structures contain levulinic acid bound in a fashion analogous to the Schiff base (Protein Database codes 1B4K, 1YLV, and 1B4E), and the actual Schiff base involving ALA has been observed by 13 C and 15 N NMR for a chemically mo...

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“…The hemB gene, which encodes b-aminolevulinic dehydratase, has been cloned and sequenced (8,9). A clone of hemB, pJL2 (kindly supplied by S. Cosloy of The City College of The City University of New York), when introduced into Oxys-6, restored its ability to grow aerobically.…”

Section: Resultsmentioning

confidence: 99%

“…The molecular structure and some of the functions of this locus have been established by other investigators (4,8,9,13). It is known that hemB controls the synthesis of 5-aminolevulinate dehydratase, an enzyme required for the dimerization of 5-aminolevulinic acid to form the monopyrolle, porphobilinogen.…”

mentioning

confidence: 89%

Oxygen sensitivity of an Escherichia coli mutant

1992

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Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media.

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The structure of the Escherichia coli hemB gene

Cited by 51 publications

References 20 publications

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

Oxygen sensitivity of an Escherichia coli mutant

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