The Zinc and Iron Binuclear Transport Center of ZupT, a ZIP Transporter from Escherichia coli.

ZupT fromEscherichia coliis a member of the Zrt-/Irt-like Protein (ZIP) transporter family, which is responsible for zinc uptake during zinc-sufficient conditions. ZIP transporters have been shown to transport different divalent metal ions including zinc, iron, manganese, and cadmium. In this study, we show that ZupT has an asymmetric binuclear metal center in the transmembrane domain; one metal-binding site, M1, binds zinc, cadmium, and iron, while the other, M2, binds iron only and with higher affinity than M1. Using site-specific mutagenesis and transport activity measurements in whole cells and proteoliposomes, we show that zinc is transported from M1, while iron is transported from M2. The two sites share a common bridging ligand, a conserved glutamate residue. M1 and M2 have ligands from highly conserved motifs in transmembrane domains 4 and 5. Additionally, M2 has a ligand from transmembrane domain 6, a glutamate residue, which is conserved in the gufA subfamily of ZIP transporters, including ZupT and the human ZIP11. Unlike cadmium, iron transport from M2 does not inhibit the zinc transport activity but slightly stimulates it. This stimulation of activity is mediated through the bridging carboxylate ligand. The binuclear zinc-iron binding center in ZupT has likely evolved to enable the transport of essential metals from two different sites without competition; a similar mechanism of metal transport is likely to be found in the gufA subfamily of ZIP transporter proteins.

Structural Role of the First Four Transmembrane Helices in ZntA, a P1B-Type ATPase from Escherichia coli.

ZntA from Escherichia coli confers resistance to toxic concentrations of Pb2+, Zn2+, and Cd2+. It is a member of the P1B-ATPase transporter superfamily, which includes the human Cu+-transporting proteins ATP7A and ATP7B. P1B-type ATPases typically have a hydrophilic N-terminal metal-binding domain and eight transmembrane helices. A splice variant of ATP7B was reported, which has 100-fold higher night-specific expression in the pineal gland; it lacks the entire N-terminal domain and the first four transmembrane helices. Here, we report our findings with Δ231-ZntA, a similar truncation we created in ZntA. Δ231-ZntA has no in vivo and greatly reduced in vitro activity. It binds one metal ion per dimer at the transmembrane site, with a 15-19000-fold higher binding affinity, indicating highly significant changes in the dimer structure of Δ231-ZntA relative to that of ZntA. Cd2+ has the highest affinity for Δ231-ZntA, in contrast to ZntA, which has the highest affinity for Pb2+. Site-specific mutagenesis of the metal-binding residues, 392Cys, 394Cys, and 714Asp, showed that there is considerable flexibility at the metal-binding site, with any two of these three residues able to bind Zn2+ and Pb2+ unlike in ZntA. However, Cd2+ binds to only 392Cys and 714Asp, with 394Cys not involved in Cd2+ binding. Three-dimensional homology models show that there is a dramatic difference between the ZntA and Δ231-ZntA dimer structures, which help to explain these observations. Therefore, the first four transmembrane helices in ZntA and P1B-type ATPases play an important role in maintaining the correct dimer structure.

Discovering proteasomal deubiquitinating enzyme inhibitors for cancer therapy: lessons from rational design, nature and old drug reposition.

The ubiquitin proteasome system has been validated as a target of cancer therapies evident by the US FDA approval of anticancer 20S proteasome inhibitors. Deubiquitinating enzymes (DUBs), an essential component of the ubiquitin proteasome system, regulate cellular processes through the removal of ubiquitin from ubiquitinated-tagged proteins. The deubiquitination process has been linked with cancer and other pathologies. As such, the study of proteasomal DUBs and their inhibitors has garnered interest as a novel strategy to improve current cancer therapies, especially for cancers resistant to 20S proteasome inhibitors. This article reviews proteasomal DUB inhibitors in the context of: discovery through rational design approach, discovery from searching natural products and discovery from repurposing old drugs, and offers a future perspective.

Association between cadmium and androgen receptor protein expression differs in prostate tumors of African American and European American men.

Cadmium is a known carcinogen that has been implicated in prostate cancer, but how it affects prostate carcinogenesis in humans remains unclear. Evidence from basic science suggests that cadmium can bind to the androgen receptor causing endocrine disruption. The androgen receptor is required for normal prostate development and is the key driver of prostate cancer progression. In this study, we examined the association between cadmium content and androgen receptor protein expression in prostate cancer tissue of African American (N = 22) and European American (N = 30) men. Although neither overall tumor cadmium content (log transformed) nor androgen receptor protein expression level differed by race, we observed a race-cadmium interaction with regard to androgen receptor expression (P = 0.003) even after accounting for age at prostatectomy, smoking history, and Gleason score. African American men had a significant positive correlation between tumor tissue cadmium content and androgen receptor expression (Pearson correlation = 0.52, P = 0.013), while European Americans showed a non-significant negative correlation between the two (Pearson correlation = -0.19, P = 0.31). These results were unchanged after further accounting for tissue zinc content or dietary zinc or selenium intake. African American cases with high-cadmium content (>median) in tumor tissue had more than double the androgen receptor expression (0.021 vs. 0.008, P = 0.014) of African American men with low-cadmium level. No difference in androgen receptor expression was observed in European Americans by cadmium level (high 0.015 vs. low 0.011, P = 0.30). Larger studies are needed to confirm these results and if upheld, determine the biologic mechanism by which cadmium increases androgen receptor protein expression in a race-dependent manner. Our results suggest that cadmium may play a role in race disparities observed in prostate cancer.

Involvement of ALAD-20S Proteasome Complexes in Ubiquitination and Acetylation of Proteasomal α2 Subunits.

The ubiquitin-proteasome pathway has gained attention as a potential chemotherapeutic target, owing to its importance in the maintenance of protein homeostasis and the observation that cancer cells are more dependent on this pathway than normal cells. Additionally, inhibition of histone deacetylases (HDACs) by their inhibitors like Vorinostat (SAHA) has also proven a useful strategy in cancer therapy and the concomitant use of proteasome and HDAC inhibitors has been shown to be superior to either treatment alone. It has also been reported that delta-aminolevulinic acid dehydratase (ALAD) is a proteasome-associated protein, and may function as an endogenous proteasome inhibitor. While the role of ALAD in the heme biosynthetic pathway is well characterized, little is known about its interaction with, and the mechanism by which it inhibits, the proteasome. In the present study, this ALAD-proteasome complex was further characterized in cultured prostate cancer cells and the effects of SAHA treatment on the regulation of ALAD were investigated. ALAD interacts with the 20S proteasomal core, but not the 19S regulatory cap. Some ubiquitinated species were detected in ALAD immunoprecipitates that have similar molecular weights to ubiquitinated proteasomal α2 subunits, suggesting preferred binding of ALAD to ubiquitinated α2. Additionally, SAHA treatment increases levels of ALAD protein and an acetylated protein with a molecular weight similar to the ubiquitinated α2 subunit. Thus, the results of this study suggest that ALAD may play a regulatory role in a previously unreported post-translational modification of proteasomal α subunits.

Gene-environment interactions between JAZF1 and occupational and household lead exposure in prostate cancer among African American men.

PURPOSE: A single nucleotide polymorphism, rs10486567, in JAZF1 has consistently been associated with increased risk of prostate cancer. The physical interaction of zinc finger proteins, such as JAZF1, with heavy metals may play a role in carcinogenesis. This study assessed potential gene-environment statistical interactions (G×E) between rs10486567 and heavy metals in prostate cancer. METHODS: In a case-only study of 228 African American prostate cancer cases, G×E between rs10486567 and sources of cadmium and lead (Pb) were assessed. Unconditional logistic regression was used to estimate interaction odds ratios (IORs), and generalized estimating equations were used for models containing nested data. Case-control validation of IORs was performed, using 82 controls frequency matched to cases on age-race. RESULTS: Among cases, a potential G×E interaction was observed between rs10486567 CC genotype and living in a Census tract with a high proportion of housing built before 1950, a proxy for household Pb exposure, when compared to CT or TT carriers (OR 1.81; 95% CI 1.04-3.16; p = 0.036). A stronger G×E interaction was observed when both housing and occupational Pb exposure were taken into account (OR 2.62; 95% CI 1.03-6.68; p = 0.04). Case-control stratified analyses showed the odds of being a CC carrier were higher in cases compared to controls among men living in areas with older housing (OR 2.03; CI 0.99-4.19; p = 0.05) or having high occupational Pb exposure (OR 2.50; CI 1.01-6.18; p = 0.05). CONCLUSIONS: In African American men, the association between JAZF1 rs10486567 and prostate cancer may be modified by exposure to heavy metals such as Pb.

Case-only gene-environment interaction between ALAD tagSNPs and occupational lead exposure in prostate cancer.

BACKGROUND: Black men have historically had higher blood lead levels than white men in the U.S. and have the highest incidence of prostate cancer in the world. Inorganic lead has been classified as a probable human carcinogen. Lead (Pb) inhibits delta-aminolevulinic acid dehydratase (ALAD), a gene recently implicated in other genitourinary cancers. The ALAD enzyme is involved in the second step of heme biosynthesis and is an endogenous inhibitor of the 26S proteasome, a master system for protein degradation and a current target of cancer therapy. METHODS: Using a case-only study design, we assessed potential gene-environment (G × E) interactions between lifetime occupational Pb exposure and 11 tagSNPs within ALAD in black (N = 260) and white (N = 343) prostate cancer cases. RESULTS: Two ALAD tagSNPs in high linkage disequilibrium showed significant interaction with high Pb exposure among black cases (rs818684 interaction odds ratio or IOR = 2.73, 95% CI 1.43-5.22, P = 0.002; rs818689 IOR = 2.20, 95% CI 1.15-4.21, P = 0.017) and an additional tagSNP, rs2761016, showed G × E interaction with low Pb exposure (IOR = 2.08, 95% CI 1.13-3.84, P = 0.019). Further, the variant allele of rs818684 was associated with a higher Gleason grade in those with high Pb exposure among both blacks (OR 3.96, 95% CI 1.01-15.46, P = 0.048) and whites (OR 2.95, 95% CI 1.18-7.39, P = 0.020). CONCLUSIONS: Genetic variation in ALAD may modify associations between Pb and prostate cancer. Additional studies of ALAD, Pb, and prostate cancer are warranted and should include black men. Prostate 74:637-646, 2014. © 2014 Wiley Periodicals, Inc.

Prostate tissue metal levels and prostate cancer recurrence in smokers.

Although smoking is not associated with prostate cancer risk overall, smoking is associated with prostate cancer recurrence and mortality. Increased cadmium (Cd) exposure from smoking may play a role in progression of the disease. In this study, inductively coupled plasma mass spectrometry was used to determine Cd, arsenic (As), lead (Pb), and zinc (Zn) levels in formalin-fixed paraffin embedded tumor and tumor-adjacent non-neoplastic tissue of never- and ever-smokers with prostate cancer. In smokers, metal levels were also evaluated with regard to biochemical and distant recurrence of disease. Smokers (N = 25) had significantly higher Cd (median ppb, p = 0.03) and lower Zn (p = 0.002) in non-neoplastic tissue than never-smokers (N = 21). Metal levels were not significantly different in tumor tissue of smokers and non-smokers. Among smokers, Cd level did not differ by recurrence status. However, the ratio of Cd ppb to Pb ppb was significantly higher in both tumor and adjacent tissue of cases with distant recurrence when compared with cases without distant recurrence (tumor tissue Cd/Pb, 6.36 vs. 1.19, p = 0.009, adjacent non-neoplastic tissue Cd/Pb, 6.36 vs. 1.02, p = 0.038). Tissue Zn levels were also higher in smokers with distant recurrence (tumor, p = 0.039 and adjacent non-neoplastic, p = 0.028). These initial findings suggest that prostate tissue metal levels may differ in smokers with and without recurrence. If these findings are confirmed in larger studies, additional work will be needed to determine whether variations in metal levels are drivers of disease progression or are simply passengers of the disease process.

From bortezomib to other inhibitors of the proteasome and beyond.

The cancer drug discovery field has placed much emphasis on the identification of novel and cancer-specific molecular targets. A rich source of such targets for the design of novel anti-tumor agents is the ubiqutin-proteasome system (UP-S), a tightly regulated, highly specific pathway responsible for the vast majority of protein turnover within the cell. Because of its critical role in almost all cell processes that ensure normal cellular function, its inhibition at one point in time was deemed non-specific and therefore not worth further investigation as a molecular drug target. However, today the proteasome is one of the most promising anti-cancer drug targets of the century. The discovery that tumor cells are in fact more sensitive to proteasome inhibitors than normal cells indeed paved the way for the design of its inhibitors. Such efforts have led to bortezomib, the first FDA approved proteasome inhibitor now used as a frontline treatment for newly diagnosed multiple myeloma (MM), relapsed/refractory MM and mantle cell lymphoma. Though successful in improving clinical outcomes for patients with hematological malignancies, relapse often occurs in those who initially responded to bortezomib. Therefore, the acquisition of bortezomib resistance is a major issue with its therapy. Furthermore, some neuro-toxicities have been associated with bortezomib treatment and its efficacy in solid tumors is lacking. These observations have encouraged researchers to pursue the next generation of proteasome inhibitors, which would ideally overcome bortezomib resistance, have reduced toxicities and a broader range of anti-cancer activity. This review summarizes the success and limitations of bortezomib, and describes recent advances in the field, including, and most notably, the most recent FDA approval of carfilzomib in July, 2012, a second generation proteasome inhibitor. Other proteasome inhibitors currently in clinical trials and those that are currently experimental grade will also be discussed.

Association of metals and proteasome activity in erythrocytes of prostate cancer patients and controls.

Information is lacking on the effects toxic environmental metals may have on the 26S proteasome. The proteasome is a primary vehicle for selective degradation of damaged proteins in a cell and due to its role in cell proliferation, inhibition of the proteasome has become a target for cancer therapy. Metals are essential to the proteasome's normal function and have been used within proteasome-inhibiting complexes for cancer therapy. This study evaluated the association of erythrocyte metal levels and proteasome chymotrypsin-like (CT-like) activity in age- and race-matched prostate cancer cases (n=61) and controls (n=61). Erythrocyte metals were measured by inductively coupled plasma mass spectrometry (ICP-MS). CT-like activity was measured by proteasome activity assay using a fluorogenic peptide substrate. Among cases, significant correlations between individual toxic metals were observed (r(arsenic-cadmium)=0.49, p<0.001; r(arsenic-lead)=0.26, p=0.04, r(cadmium-lead) 0.53, p<0.001), but there were no significant associations between metals and CT-like activity. In contrast, within controls there were no significant associations between metals, however, copper and lead levels were significantly associated with CT-like activity. The associations between copper and lead and proteasome activity (r(copper-CT-like)=-0.28, p=0.002 ; r(lead-CT-like)=0.23, p=0.011) remained significant in multivariable models that included all of the metals. These findings suggest that biologically essential metals and toxic metals may affect proteasome activity in healthy controls and, further, show that prostate cancer cases and controls differ in associations between metals and proteasome activity in erythrocytes. More research on toxic metals and the proteasome in prostate cancer is warranted.

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity.

ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved 392CPC394 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue.

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.

Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity.

ZntA, a P1B-type ATPase, confers resistance specifically to Pb2+, Zn2+, and Cd2 in Escherichia coli. Inductively coupled plasma mass spectrometry measurements show that ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain. Both sites can bind monovalent and divalent metal ions. Two proteins, deltaN-ZntA, in which the N-terminal domain is deleted, and C59A/C62A-ZntA, in which the N-terminal metal-binding site is disabled by site-specific mutagenesis, can only bind one metal ion. Because C59A/C62A-ZntA can bind a metal ion at the transmembrane site, the N-terminal domain does not block direct access of metal ions to it from the cytosol. A third mutant protein, C392A/C394A-ZntA, in which cysteines from the conserved CPC motif in transmembrane helix 6 are altered, binds metal ions only at the N-terminal site, indicating that both these cysteines form part of the transmembrane site. The metal affinity of the transmembrane site was determined in deltaN-ZntA and C59A/C62A-ZntA by competition titration using a metal ion indicator and by tryptophan fluorescence quenching. The binding affinity for the physiological substrates, Zn2+, Pb2+, and Cd2+, as well as for the extremely poor substrates, Cu2+, Ni2+, and Co2+, range from 10(6)-10(10) M(-1), and does not correlate with the metal selectivity shown by ZntA. Selectivity in ZntA possibly results from differences in metal-binding geometry that produce different structural responses. The affinity of the transmembrane site for metal ions is of similar magnitude to that of the N-terminal site [Liu J. et al. (2005) Biochemistry 44, 5159-5167]; thus, metal transfer between them would be facile.

Kinetic analysis of metal binding to the amino-terminal domain of ZntA by monitoring metal-thiolate charge-transfer complexes.

ZntA, a P(1B)-ATPase transporter from Escherichia coli, mediates resistance specifically to Pb(2+), Zn(2+), and Cd(2+) by active efflux. ZntA has a hydrophilic N-terminal domain that binds one metal ion. This domain, approximately 120 residues long, contains the GXXCXXC motif that has been shown to be the binding site for metal ions such as Cu(+) and Zn(2+) in P(1B)-type ATPases, and an additional cysteine-rich motif, CCCDGAC. We report here that binding of Pb(2+) and Cd(2+) to this domain produces changes in the absorbance spectrum in the 250-400 nm range indicative of metal-thiolate charge-transfer complexes. The spectral changes indicate that only two cysteines are ligands to Cd(2+), but three or more cysteines are involved in binding Pb(2+); this confirms earlier results that the GXXCXXC sequence is not sufficient to bind Pb(2+), which likely involves residues from the CCCDGAC motif. The absorbance changes were used to measure metal binding kinetics of the N-terminal domain using stopped-flow techniques. Binding was described by simple second-order kinetics with a rate constant, k(on), of approximately 10(6)-10(7) M(-)(1) s(-)(1), at 4 degrees C. The activation energy of binding is similar for both Pb(2+) and Cd(2+); however, the entropy change is greater for Pb(2+). The surprisingly large rate constant for metal binding to the N-terminal domain of ZntA, compared to its low turnover rate, indicates that this step is not rate limiting in the overall transport mechanism. These results, in conjunction with earlier studies, suggest that metal binding to the transmembrane site in ZntA or metal release from the transporter is the slow step in the reaction cycle.

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc.

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).

Role of glycine 81 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate specificity and oxidase activity.

(S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Despite a high degree of sequence and structural similarity, this family can be divided into three subgroups based on the different oxidants utilized in the second oxidative half-reaction. Only the oxidases show high reactivity with molecular oxygen. Structural data indicate that the relative position of a peptide loop and the isoalloxazine ring of the FMN is slightly different in the oxidases compared to the dehydrogenases; the last residue on this loop is either an alanine or glycine. We examined the effect of the G81A, G81S, G81V, and G81D mutations in MDH on the overall reaction and especially on the suppression of activity with oxygen. G81A had a higher specificity for small substrates compared to that of wtMDH, though the affinity for (S)-mandelate was relatively unchanged. The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Redox-potential measurements indicate that the reduction in activity is due to the decrease in electrophilicity of the FMN. The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. These results suggest that one of the ways the oxidase activity of MDH is controlled is through steric effects because of the relative positions of the FMN and the Gly81 loop.

Esters of mandelic acid as substrates for (S)-mandelate dehydrogenase from Pseudomonas putida: implications for the reaction mechanism.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes (S)-mandelate to benzoylformate. In this work, we show that the ethyl and methyl esters of (S)-mandelic acid are substrates for MDH. Although the binding affinity of the neutral esters is 25-50-fold lower relative to the negatively charged (S)-mandelate, they are oxidized with comparable k(cat)s. Substrate analogues in which the carbonyl group on the C-1 carbon is replaced by other electron-withdrawing groups were not substrates. The requirement of a carbonyl group on the C-1 carbon in a substrate suggests that the negative charge developed during the reaction is stabilized by delocalization to the carbonyl oxygen. Arg277, a residue that is important in both binding and transition state stabilization for the activity with (S)-mandelate, is also critical for transition state stabilization for the esters, but not for their binding affinity. We previously showed that the substrate oxidation half-reaction with (S)-mandelate has two rate-limiting steps of similar activation energies and proceeds through the formation of a charge-transfer complex of an electron-rich donor and oxidized FMN [Dewanti, A. R., and Mitra, B. (2003) Biochemistry 42, 12893-12901]. This charge-transfer intermediate was observed with the neutral esters as well. The observation of this electron-rich intermediate for the oxidation of an uncharged substrate to an uncharged product, as well as the critical role of Arg277 in the reaction with the esters, provides further evidence that the MDH reaction mechanism is not a concerted transfer of a hydride ion from the substrate to the FMN, but involves the transient formation of a carbanion/ene(di)olate intermediate.

High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (S)-mandelate dehydrogenase.

The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-A resolution. The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176-195 of glycolate oxidase from spinach. Both structures permit full tracing of the polypeptide backbone chain from residues 4-356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 A resolution. The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction. The only other structural changes within the protein interior are a 10 degrees rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 A. Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme. The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group. Implications for the low oxidase activity of the reduced enzyme are also discussed.

A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida.

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.