Protein Engineering of P450BM3 by Rational Redesign

The potential of flavocytochrome P450BM3 (CYP102A1) from Bacillus megaterium for industrial chemical transformation and biotechnological application is widely acknowledged. The crystal structures of P450BM3 with fatty acid substrates bound present non-productive modes of binding of substrate with their carbons distant from the iron and the ω-terminal end in a hydrophobic pocket at one side of the active site. Comparison between substrate-free and substrate-bound structures of P450BM3 revealed two pockets (A-arm and B-arm) in the substrate binding channel. In this thesis, A82(I/F/W) mutants in which the ‘B-arm’ pocket is filled by large hydrophobic side chains at position 82 were constructed and characterised. The A82F and A82W mutants have greater affinities for substrates (~ 800-fold) as well as being more effective catalysts of indole hydroxylation than the wild-type enzyme. The crystal structure of the haem domain of the A82F mutant with bound palmitate showed different substrate binding position, in which the substrate is closer to the haem iron than wild-type enzyme. On this basis, a second series of mutants with substitutions at position 438 as well as 82, in which the ‘A-arm’ pocket is modulated by large hydrophobic side chains, were constructed and characterised. The hydroxylation of 11-methyllaurate by wild-type was found to yield traces of the ω-hydroxylated product, which is the first observation of ω-hydroxylase activity of wild-type P450BM3 to date. The mutants with both ‘B-arm’ pocket and ‘A-arm’ pocket filled with larger hydrophobic residues (A82F-T438(V/I/L/F) mutants) demonstrated 2- to 3-fold increases in the formation of ω-hydroxyl-11-methyllaurate. Notably, the A82F-T438L and A82F-T438F mutants also presented a marked enhancement of stereo-selectivity for styrene epoxidation to generate R-styrene oxide (~ 30-fold), suggesting that not only that these mutants of P450BM3 will be valuable catalysts for synthetically useful hydroxylation reactions but also that structure-based rational redesign will be one of the most efficient tools to generate novel biocatalysts

Nonparametric estimation and test of conditional Kendall's tau under semi-competing risks data and truncated data

<div><p>In this article, we focus on estimation and test of conditional Kendall's tau under semi-competing risks data and truncated data. We apply the inverse probability censoring weighted technique to construct an estimator of conditional Kendall's tau, </p><p></p><p></p><p><mi>τ</mi><mi>c</mi></p><p></p><p></p>. Then, this study provides a test statistic for <p></p><p></p><p><mi>H</mi><mn>0</mn></p><mo>:</mo><p><mi>τ</mi><mi>c</mi></p><mo>=</mo><p><mi>τ</mi><mn>0</mn></p><p></p><p></p>, where <p></p><p></p><p><mi>τ</mi><mn>0</mn></p><mo>∈</mo><mo stretchy="false">(</mo><mo>−</mo><mn>1</mn><mo>,</mo><mn>1</mn><mo stretchy="false">)</mo><p></p><p></p>. When two random variables are quasi-independent, it implies <p></p><p></p><p><mi>τ</mi><mi>c</mi></p><mo>=</mo><mn>0</mn><p></p><p></p>. Thus, <p></p><p></p><p><mi>H</mi><mn>0</mn></p><mo>:</mo><p><mi>τ</mi><mi>c</mi></p><mo>=</mo><mn>0</mn><p></p><p></p> is a proxy for quasi-independence. Tsai [<a href="#CIT0012" target="_blank">12</a>], and Martin and Betensky [<a href="#CIT0010" target="_blank">10</a>] considered the testing problem for quasi-independence. Via simulation studies, we compare the three test statistics for quasi-independence, and examine the finite-sample performance of the proposed estimator and the suggested test statistic. Furthermore, we provide the large sample properties for our proposed estimator. Finally, we provide two real data examples for illustration.<p></p></div

Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase

NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this

Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency

Cytochrome P450BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase, is a member of a very large superfamily of monooxygenase enzymes. The available crystal structures of the enzyme show non-productive binding of substrates with their ω-end distant from the iron in a hydrophobic pocket at one side of the active site. We have constructed and characterised mutants in which this pocket is filled by large hydrophobic sidechains replacing alanine at position 82. The mutants having phenylalanine or tryptophan at this position have very much (~800-fold) greater affinity for substrate, with a greater conversion of the haem iron to the high-spin state, and similarly increased catalytic efficiency. The enzyme as isolated contains bound palmitate, reflecting this much higher affinity. We have determined the crystal structure of the haem domain of the Ala82Phe mutant with bound palmitate; this shows that the substrate is binding differently from the wild-type enzyme but still distant from the haem iron. Detailed analysis of the structure indicates that the tighter binding in the mutant reflects a shift in the conformational equilibrium of the substrate-free enzyme towards the conformation seen in the substrate complex rather than differences in the enzyme-substrate interactions. On this basis, we outline a sequence of events for the initial stages of the catalytic cycle. The Ala82Phe and Ala82Trp mutants are also very much more effective catalysts of indole hydroxylation than the wild-type enzyme, suggesting that they will be valuable starting points for the design of mutants to catalyse synthetically useful hydroxylation reactions

Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation

The vast majority of <i>in vitro</i> structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an <i>in vivo</i> regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated <i>in vivo</i>. Our results show that, while Rad53 can display active conformations under various conditions, simulation of <i>in vivo</i> regulatory conditions confers functionally relevant autophosphorylation