Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues

Püttner, Irene B.; Sarkar, Hemanta K.; Padan, Etana; Lolkema, Julius S.; Kaback, H. Ronald

doi:10.1021/bi00432a027

Cited by 74 publications

(44 citation statements)

References 42 publications

(84 reference statements)

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“…Importantly, as evidenced by binding studies with NPG, E325A permease binds the high-affinity ligand with a KD approximating that of wild-type permease. The finding is consistent with the observation that counterflow, a process that exhibits an apparent K, similar to that observed for active transport, is intact in the mutant but is in marked contrast to findings with the R302L and H322R permeases which exhibit dramatically decreased affinities (D. R. Menick, L. Patel, and H. R. Kaback, unpublished information;Puttner et al, 1989). Therefore, in addition to postulating that His322 may be a component of the substrate binding site as well as an intermediate in H+ translocation (Wttner et al, 1989), it is tempting to speculate further that a protonated imidazole at position 322 may be required for high-affinity binding.…”

Section: Discussionsupporting

confidence: 88%

“…x e preceding paper (Puttner et al, 1989) confirms and extends earlier observations (Padan et al, 1979(Padan et al, , 1985Patel et al, 1982;Garcia et al, 1982;Piittner et al, 1986) focusing on the importance of His322 in lactose/H+ symport by the lac permease. Thus, evidence was presented indicating that His322 may be directly involved in lactose-coupled H+ translocation.…”

supporting

confidence: 86%

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Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Carrasco¹,

Püttner²,

Antes³

et al. 1989

Biochemistry

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105

147

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Section: Discussionsupporting

confidence: 88%

supporting

confidence: 86%

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Carrasco¹,

Püttner²,

Antes³

et al. 1989

Biochemistry

Self Cite

105

147

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“…This amino acid residue has been found to play an important role in the transport of lactose (11,24). The protein contains only one cysteine residue, at position 312.…”

Section: Methodsmentioning

confidence: 99%

Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved

1991

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The genes coding for the lactose permease and 0-galactosidase, two proteins involved in the metabolism of lactose by LactobaciUlus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli. The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs). One of these ORFs codes for the lactose permease gene, and the other codes for the P-galactosidase gene. The lactose permease gene is located in front of the I-galactosidase gene, with 3 bp in the intergenic region. The two genes are probably transcribed as one operon. Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the ,-galactosidase gene. This promoter is similar to those found in E. coli with general characteristics of GC-rich organisms. In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L. bulgaricus genome, which is rich in GC (GC content of 54%). The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus. The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E. coli. Little homology was found between the lactose permease of L. bulgaricus and E. coli, but the residues which are involved in the binding and the transport of lactose are conserved. The carboxy terminus is similar to that of the enzyme Ill of several phosphoenolpyruvate-dependent phosphotransferase systems.Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are dairy lactic acid bacteria that are widely used in the fermentation of milk (25). During their growth in milk, lactose is used as the primary energy source. Two systems for transport and metabolism of lactose are known in lactic acid bacteria: (i) a phosphoenolpyruvate (PEP) lactose phosphotransferase system (PTS) with a phospho-p-galactosidase enzyme (21) and (ii) a lactose permease system with a P-galactosidase. The PEP-PTS system is found in many species, including lactococci (reviewed in references 30 and 31).L. bulgaricus utilizes lactose via the second pathway. Lactose is brought into the cell as the free sugar and cleaved by P-galactosidase. The glucose moiety of the sugar is further metabolized while the galactose moiety is released (8, 23). The lactose operon of Escherichia coli, which also has a lactose permease (lacY) and 3-galactosidase (lacZ), has been extensively studied, and the proteins involved are being characterized (1-4, 11, 13, 24). Not much is known, however, about the system in lactic acid bacteria. Recently, there has been a report on the cloning and sequencing of the ,-galactosidase gene from L. bulgaricus (28). This group found that ,B-galactosidase from L. bulgaricus has an average similarity of 34% to that from E. coli, with stretches of high homology around the regions involved in activit...

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“…A great deal of work has been directed towards the role of His-322 in LacY [11,12]. This residue has been implicated in lactose-coupled H ÷ translocation and forms part of a putative proton relay [245][246][247][248][249]. The proton or charge relay mechanism assumes a role for His-322, Glu-325 (which should be on the same side of a-helix X as His-322), and Arg-302 (putative helix IX) in proton translocation, which is based on analogies with proton transfer via Asp, His and Ser in serine-type proteinases [250].…”

Section: Vii-a Histidine Residuesmentioning

confidence: 99%

Secondary solute transport in bacteria

Poolman

Konings

1993

Biochimica et Biophysica Acta (BBA) - Bioenergetics

202

160

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Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues

Cited by 74 publications

References 42 publications

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved

Secondary solute transport in bacteria

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